Vitamin receptor binding drug delivery conjugates

ABSTRACT

The invention describes a vitamin receptor binding drug delivery conjugate, and preparations therefor. The drug delivery conjugate consists of a vitamin receptor binding moiety, a bivalent linker (L), and a drug. The vitamin receptor binding moiety includes vitamins, and vitamin receptor binding analogs and derivatives thereof, and the drug includes analogs and derivatives thereof. The vitamin receptor binding moiety is covalently linked to the bivalent linker, and the drug, or the analog or the derivative thereof, is covalently linked to the bivalent linker, wherein the bivalent linker (L) includes components such as spacer linkers, releasable linkers, and heteroatom linkers, and combinations thereof. Methods and pharmaceutical compositions for eliminating pathogenic cell populations using the drug delivery conjugate are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. PatentApplication No. 60/442,845, entitled “Vitamin-Receptor Binding DrugDelivery Conjugates,” filed Jan. 27, 2003, U.S. Patent Application No.60/492,119, entitled “Vitamin-Receptor Binding Drug DeliveryConjugates,” filed Aug. 1, 2003, and U.S. Patent Application No.60/516,188, entitled “Vitamin-Receptor Binding Drug DeliveryConjugates,” filed Oct. 31, 2003. The entirety of the disclosures ofeach of these applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for use intargeted drug delivery. More particularly, the invention is directed tovitamin receptor binding drug delivery conjugates for use in treatingdisease states caused by pathogenic cell populations and to a method andpharmaceutical composition therefor.

BACKGROUND

The mammalian immune system provides a means for the recognition andelimination of tumor cells, other pathogenic cells, and invading foreignpathogens. While the immune system normally provides a strong line ofdefense, there are many instances where cancer cells, other pathogeniccells, or infectious agents evade a host immune response and proliferateor persist with concomitant host pathogenicity. Chemotherapeutic agentsand radiation therapies have been developed to eliminate, for example,replicating neoplasms. However, many of the currently availablechemotherapeutic agents and radiation therapy regimens have adverse sideeffects because they work not only to destroy pathogenic cells, but theyalso affect normal host cells, such as cells of the hematopoieticsystem. The adverse side effects of these anticancer drugs highlight theneed for the development of new therapies selective for pathogenic cellpopulations and with reduced host toxicity.

Researchers have developed therapeutic protocols for destroyingpathogenic cells by targeting cytotoxic compounds to such cells. Many ofthese protocols utilize toxins conjugated to antibodies that bind toantigens unique to or overexpressed by the pathogenic cells in anattempt to minimize delivery of the toxin to normal cells. Using thisapproach, certain inmnunotoxins have been developed consisting ofantibodies directed to specific antigens on pathogenic cells, theantibodies being linked to toxins such as ricin, Pseudomonas exotoxin,Diptheria toxin, and tumor necrosis factor. These inmunotoxins targetpathogenic cells, such as tumor cells, bearing the specific antigensrecognized by the antibody (Olsnes, S., Immunol. Today, 10, pp. 291-295,1989; Melby, E. L., Cancer Res., 53(8), pp. 1755-1760, 1993; Better, M.D., PCT Publication Number WO 91/07418, published May 30, 1991).

Another approach for targeting populations of pathogenic cells, such ascancer cells or foreign pathogens, in a host is to enhance the hostimmune response against the pathogenic cells to avoid the need foradministration of compounds that may also exhibit independent hosttoxicity. One reported strategy for immunotherapy is to bind antibodies,for example, genetically engineered multimeric antibodies, to thesurface of tumor cells to display the constant region of the antibodieson the cell surface and thereby induce tumor cell killing by variousimmune-system mediated processes (De Vita, V. T., Biologic Therapy ofCancer, 2d ed. Philadelphia, Lippincott, 1995; Soulillou, J. P., U.S.Pat. No. 5,672,486). However, these approaches have been complicated bythe difficulties in defining tumor-specific antigens.

SUMMARY OF THE INVENTION

In an attempt to develop effective therapies specific for pathogeniccells and with minimized toxicity for normal cells, vitamin receptorbinding drug delivery conjugates have been developed. The presentinvention is applicable to populations of pathogenic cells that cause avariety of pathologies in host animals. The pathogenic cells that can betreated with the drug delivery conjugates of the present inventioninclude tumor cells, infectious agents such as bacteria and viruses,bacteria- or virus-infected cells, and any other type of pathogeniccells that uniquely express, preferentially express, or overexpressvitamin receptors or receptors that bind vitamin analogs or derivatives.

In one embodiment there is provided a vitamin receptor binding drugdelivery conjugate. The drug delivery conjugate comprises a vitaminreceptor binding moiety, a bivalent linker, and a drug. As used herein,“V” refers to a vitamin receptor binding moiety and includes vitamins,and vitamin receptor binding analogs or derivatives thereof, and theterm “vitamin or analog or derivative thereof” refers to vitamins andanalogs and derivatives thereof that are capable of binding vitaminreceptors. As used herein, “D” refers to drugs and includes analogs orderivatives thereof. The vitamin, or the analog or the derivativethereof, is covalently bound to the bivalent linker (L), and the drug,or the analog or the derivative thereof, is also covalently bound to thebivalent linker (L). The bivalent linker (L) can comprise multiplelinkers. For example, the bivalent linker (L) can comprise one or morecomponents selected from spacer linkers (l_(s)), releasable linkers(l_(r)), and heteroatom linkers (l_(H)), and combinations thereof, inany order.

Drug delivery conjugates that illustrate this embodiment include: V-L-DV-(l_(r))_(c)-D V-(l_(s))_(a)-D V-(l_(s))_(a)-(l_(r))_(c)-DV-(l_(r))_(c)-(l_(s))_(a)-D V-(l_(H))_(b)-(l_(r))_(c)-DV-(l_(r))_(c)-(l_(H))_(b)-D V-(l_(H))_(d)-(l_(r))_(c)-(l_(H))_(e)-DV-(l_(s))_(a)-(l_(H))_(b)-(l_(r))_(c)-DV-(l_(r))_(c)-(l_(H))_(b)-(l_(s))_(a)-DV-(l_(H))_(d)-(l_(s))_(a)-(l_(r))_(c)-(l_(H))_(e)-DV-(l_(H))_(d)-(l_(r))_(c)-(l_(s))_(a)-(l_(H))_(e)-DV-(l_(H))_(d)-(l_(s))_(a)-(l_(H))_(b)-(l_(r))_(c)-(l_(H))_(e)-DV-(l_(H))_(d)-(l_(r))_(c)-(l_(H))_(b)-(l_(s))_(a)-(l_(H))_(e)-DV-(l_(s))_(a)-(l_(r))_(c)-(l_(H))_(b)-DV-[(l_(s))_(a)-(l_(H))_(b)]_(d)-(l_(r))_(c)-(l_(H))_(e)-Dwherein a, b, c, d, and e are each independently 0, 1, 2, 3, or 4,(l_(s)), (l_(H)), and (l_(r)) are as defined herein, V is a vitamin, oranalog or derivative thereof, and D is a drug, or analog or derivativethereof, and where the bivalent L encompasses one or a variety of(l_(s)), (l_(H)), and (l_(r)), in any order and in any combination. Itis understood that the foregoing examples of the bivalent linker L areintended to illustrate, and not limit, the wide variety of assemblies of(l_(H)), (l_(s)), and (l_(r)) that the bivalent linker encompasses.

It is understood that each of the spacer, heteroatom, and releasablelinkers are bivalent. It should be further understood that theconnectivity between each of the various spacer, heteroatom, andreleasable linkers, and between the various spacer, heteroatom, andreleasable linkers and D and/or V, as defined herein, may occur at anyatom found in the various spacer, heteroatom, and releasable linkers,and is not necessarily at any apparent end of any of the various spacer,heteroatom, or releasable linkers. For example, in the illustrativeembodiment where the bivalent linker is:

i.e. where the bivalent linker L is -(l_(H))-(l_(s))₅-(l_(r)-l_(H))₂-D,where (l_(H)) is nitrogen, (l_(s))₅ is Ala-Glu-Lys-Asp-Asp, and(l_(r)-l_(H))₂ is —(CH₂)₂—S—S—(CH₂)₂—O—C(O)—O—, respectively, the(l_(r)-l_(H))₂ linker is connected to middle portion of the (l_(s))₅linker.

In another embodiment there is provided a vitamin receptor binding drugdelivery conjugate. The drug delivery conjugate comprises a vitaminreceptor binding moiety, a bivalent linker (L), and a drug, and thebivalent linker (L) comprises one or more heteroatom linkers (l_(H)).The vitamin receptor binding moiety is covalently bound to the bivalentlinker (L) through a first heteroatom linker (l_(H))_(d) and the drug iscovalently bound to the bivalent linker (L) through a second heteroatomlinker (l_(H))_(e). The bivalent linker (L) also comprises one or morespacer linkers and releasable linkers, wherein the spacer linkers andthe releasable linkers may be covalently linked to each other through athird heteroatom linker (l_(H))_(b). A drug delivery conjugate thatillustrates this embodiment is as follows:V-(l_(H))_(d)-(l_(s))_(a)-(l_(H))_(b)-(l_(r))_(c)-(l_(H))_(e)-Dwherein a, b, c, d, and e are each independently 0, 1, 2, 3, or 4,(l_(s)), (l_(H)), and (l_(r)), and V and D are as defined herein, andwhere the bivalent linker L encompasses (l_(s)), (l_(H)), and (l_(r)) asillustrated.

In another embodiment there is provided a vitamin receptor binding drugdelivery conjugate. The drug delivery conjugate comprises a vitaminreceptor binding moiety, a bivalent linker (L), and a drug, and thebivalent linker (L) comprises an heteroatom linker (l_(H)). The vitaminreceptor binding moiety is a vitamin, or an analog or a derivativethereof, and the drug includes analogs or derivatives thereof. Thevitamin, or the analog or the derivative thereof, is covalently bound tothe bivalent linker (L) and the drug, or the analog or the derivativethereof, is covalently bound to the bivalent linker (L). The bivalentlinker (L) also comprises spacer linkers and releasable linkers, and thespacer linkers and the releasable linkers may be covalently bound toeach other through the heteroatom linker. A drug delivery conjugate thatillustrates this embodiment is as follows:V-(l_(s))_(a)-(l_(H))_(b)-(l_(r))_(c)-Dwherein a, b, and c are each independently 0, 1, 2, 3, or 4, (l_(s)),(l_(H)), and (l_(r)), and V and D are as defined herein, and where thebivalent linker L encompasses (l_(s)), (l_(H)), and (l_(r)) asillustrated.

In another embodiment, a vitamin receptor binding drug deliveryconjugate of the general formula V-L-D is provided. In this embodiment,L is constructed from one or more linkers (l_(r))_(c), (l_(s))_(a), and(l_(H))_(b), and combinations thereof, in any order, where (l_(r)) is areleasable linker, (l_(s)) is a spacer linker, (l_(H)) is an heteroatomlinker, and a, b, and c are each independently 0, 1, 2, 3, or 4, V is avitamin, or an analog or a derivative thereof, and D is a drug, or ananalog or a derivative thereof. It is appreciated that the drug deliveryconjugates described herein may include a bivalent linker that has morethan one spacer linker, releasable linker, or heteroatom linker. Forexample, bivalent linkers that include two or more releasable linkers(l_(r)) are contemplated. Further, configurations of such releasablelinkers include bivalent linkers where the releasable linkers arecovalently linked to each other, and where the releasable linkers areseparated from each other by one or more heteroatom linkers and/orspacer linkers.

In another embodiment, a vitamin receptor binding drug deliveryconjugate of the general formula V-L-D is described, wherein L is abivalent linker comprising (l_(s))_(a) and (l_(H))_(b), and combinationsthereof in any order, wherein (l_(s))_(a) and (l_(H))_(b), and V and Dare as defined herein. In this embodiment, the drug in the drug deliveryconjugate can be a hapten such as, but not limited to, fluorescein,dinitrophenyl, and the like.

In another embodiment, a vitamin receptor binding drug deliveryconjugate of the general formula V-L-D is described, wherein L is abivalent linker comprising (l_(s))_(a), (l_(H))_(b), and (l_(r))_(c),and combinations thereof in any order, wherein (l_(s))_(a) and(l_(H))_(b), and V and D are as defined herein, and where at least oneof (l_(r)) is not a disulfide. It is appreciated that bivalent linkersin this embodiment having more than one (l_(r)), i.e. where c is greaterthan 1, may include a disulfide releasable linker in addition to anotheror other releasable linkers.

In one aspect of the various vitamin receptor binding drug deliveryconjugates described herein, the bivalent linker comprises an heteroatomlinker, a spacer linker, and a releasable linker taken together to form3-thiosuccinimid-1-ylalkyloxymethyloxy, where the methyl is optionallysubstituted with alkyl or substituted aryl.

In another aspect, the bivalent linker comprises an heteroatom linker, aspacer linker, and a releasable linker taken together to form3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl forms anacylaziridine with the drug, or analog or derivative thereof.

In another aspect, the bivalent linker comprises an heteroatom linker, aspacer linker, and a releasable linker taken together to form1-alkoxycycloalkylenoxy.

In another aspect, the bivalent linker comprises a spacer linker, anheteroatom linker, and a releasable linker taken together to formalkyleneaminocarbonyl(dicarboxylarylene)carboxylate.

In another aspect, the bivalent linker comprises a releasable linker, aspacer linker, and a releasable linker taken together to formdithioalkylcarbonylhydrazide, where the hydrazide forms an hydrazonewith the drug, or analog or derivative thereof.

In another aspect, the bivalent linker comprises an heteroatom linker, aspacer linker, and a releasable linker taken together to form3-thiosuccinimid-1-ylalkylcarbonylhydrazide, where the hydrazide formsan hydrazone with the drug, or analog or derivative thereof

In another aspect, the bivalent linker comprises an heteroatom linker, aspacer linker, an heteroatom linker, a spacer linker, and a releasablelinker taken together to form 3-thioalkylsulfonylalkyl(disubstitutedsilyl)oxy, where the disubstituted silyl is substituted with alkyl oroptionally substituted aryl.

In another aspect, the bivalent linker comprises a plurality of spacerlinkers selected from the group consisting of the naturally occurringamino acids and stereoisomers thereof.

In another aspect, the bivalent linker comprises a releasable linker, aspacer linker, and a releasable linker taken together to form3-dithioalkyloxycarbonyl, where the carbonyl forms a carbonate with thedrug, or analog or derivative thereof.

In another aspect, the bivalent linker comprises a releasable linker, aspacer linker, and a releasable linker taken together to form3-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbonate withthe drug, or analog or derivative thereof, and the aryl is optionallysubstituted.

In another aspect, the bivalent linker comprises an heteroatom linker, aspacer linker, a releasable linker, a spacer linker; and a releasablelinker taken together to form3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the alkylideneforms an hydrazone with the drug, or analog or derivative thereof, eachalkyl is independently selected, and the oxyalkyloxy is optionallysubstituted with alkyl or optionally substituted aryl.

In another aspect, the bivalent linker comprises a releasable linker, aspacer linker, and a releasable linker taken together to form3-dithioalkyloxycarbonylhydrazide.

In another aspect, the bivalent linker comprises a releasable linker, aspacer linker, and a releasable linker taken together to form3-dithioalkylamino, where the amino forms a vinylogous amide with thedrug, or analog or derivative thereof.

In another aspect, the bivalent linker comprises a releasable linker, aspacer linker, and a releasable linker taken together to form3-dithioalkylamino, where the amino forms a vinylogous amide with thedrug, or analog or derivative thereof, and the alkyl is ethyl.

In another aspect, the bivalent linker comprises a releasable linker, aspacer linker, and a releasable linker taken together to form3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate withthe drug, or analog or derivative thereof.

In another aspect, the bivalent linker comprises a releasable linker, aspacer linker, and a releasable linker taken together to form3-dithioalkylaminocarbonyl, where the carbonyl forms a carbamate withthe drug, or analog or derivative thereof, and the alkyl is ethyl.

In another aspect, the bivalent linker comprises a releasable linker, aspacer linker, and a releasable linker taken together to form3-dithioarylalkyloxycarbonyl, where the carbonyl forms a carbamate or acarbamoylaziridine with the drug, or analog or derivative thereof.

In one aspect, the releasable, spacer, and heteroatom linkers may bearranged in such a way that subsequent to the cleavage of a bond in thebivalent linker, released functional groups chemically assist thebreakage or cleavage of additional bonds, also termed anchimericassisted cleavage or breakage. An illustrative embodiment of such abivalent linker or portion thereof includes compounds having theformula:

where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is aninteger selected from 0, 1, 2, and 3, R is hydrogen, or a substituent,including a substituent capable of stabilizing a positive chargeinductively or by resonance on the aryl ring, such as alkoxy, and thelike, and the symbol (*) indicates points of attachment for additionalspacer, heteroatom, or releasable linkers forming the bivalent linker,or alternatively for attachment of the drug, or analog or derivativethereof, or the vitamin, or analog or derivative thereof It isappreciated that other substituents may be present on the aryl ring, thebenzyl carbon, the alkanoic acid, or the methylene bridge, including butnot limited to hydroxy, alkyl, alkoxy, alkylthio, halo, and the like.Assisted cleavage may include mechanisms involving benzyliumintermediates, benzyne intermediates, lactone cyclization, oxoniumintermediates, beta-elimination, and the like. It is farther appreciatedthat, in addition to fragmentation subsequent to cleavage of thereleasable linker, the initial cleavage of the releasable linker may befacilitated by an anchimerically assisted mechanism.

In another embodiment, a vitamin receptor binding drug deliveryconjugate intermediate is provided. The intermediate comprises a vitaminreceptor binding moiety, a bivalent linker, having a first end and asecond end, and a coupling group. The vitamin receptor binding moiety isa vitamin, or an analog or a derivative thereof, and the coupling groupis a nucleophile, an electrophile, or a precursor thereof The vitaminreceptor binding moiety is covalently attached to the bivalent linker atthe first end of the bivalent linker, and the coupling group iscovalently attached to the bivalent linker at the second end of thebivalent linker, and the bivalent linker comprises one or more spacerlinkers, releasable linkers, and heteroatom linkers, and combinationsthereof, in any order.

In another embodiment, a vitamin receptor binding drug deliveryconjugate intermediate is described. The intermediate comprises abivalent linker, having a first end and a second end, a drug, or ananalog or a derivative thereof, and a coupling group. The bivalentlinker comprises one or more components selected from spacer linkers,releasable linkers, and heteroatom linkers, as described herein. Thecoupling group is covalently attached to the bivalent linker at thefirst end of the bivalent linker, and the drug or analog or derivativethereof is covalently attached to the bivalent linker at the second endof the bivalent linker. Further, the coupling group is a nucleophile, anelectrophile, or a precursor thereof, capable of forming a covalent bondwith a vitamin receptor binding moiety, where the vitamin receptorbinding moiety is a vitamin, or an analog or a derivative thereof.

In another illustrative embodiment of the vitamin receptor binding drugdelivery conjugate intermediate described herein, the coupling group isa Michael acceptor, and the bivalent linker includes a releasable linkerhaving the formula —C(O)NHN═, —NHC(O)NHN═, or —CH₂C(O)NHN═. In oneillustrative aspect of the vitamin receptor binding drug deliveryconjugate intermediate described herein, the coupling group and thebivalent linker are taken together to form a compound having theformula:

or a protected derivative thereof, where D is the drug, or an analog ora derivative thereof, capable of forming a hydrazone as illustratedherein; and n is an integer such as 1, 2, 3, or 4. In anotherillustrative aspect of the vitamin receptor binding drug deliveryconjugate intermediate described herein, the vitamin, or an analog or aderivative thereof, includes an alkylthiol nucleophile.

In another illustrative embodiment of the vitamin receptor binding drugdelivery conjugate intermediate described, herein, the coupling group isa heteroatom, such as nitrogen, oxygen, or sulfur, and the bivalentlinker includes one or more heteroatom linkers and one or more spacerlinkers covalently connecting the vitamin or analog or derivativethereof to the coupling group. In one illustrative aspect, the vitaminreceptor binding drug delivery conjugate intermediate described hereinincludes a compound having the formula:

or a protected derivative thereof, where X is oxygen, nitrogen, orsulfur, and m is an integer such as 1, 2, or 3, and where V, l_(s), andl_(H) are as defined herein.

In another illustrative aspect, the vitamin receptor binding drugdelivery conjugate intermediate described herein includes a compoundhaving the formula:

or a protected derivative thereof, where X is nitrogen or sulfur, whereV and l_(s) are as defined herein.

In another illustrative aspect, the vitamin receptor binding drugdelivery conjugate intermediate described herein includes a compoundhaving the formula:

or a protected derivative thereof, where Y is hydrogen or a substituent,illustratively an electron withdrawing substituent, including but notlimited to nitro, cyano, halo, alkylsulfonyl, a carboxylic acidderivative, and the like, and where V and l_(s) are as defined herein.

In another illustrative embodiment of the vitamin receptor binding drugdelivery conjugate intermediate described herein, the coupling group isa Michael acceptor, and the bivalent linker includes one or moreheteroatom linkers and one or more spacer linkers covalently connectingthe vitamin or analog or derivative thereof to the coupling group. Inone illustrative aspect of the vitamin receptor binding drug deliveryconjugate intermediate described herein, the coupling group and thebivalent linker are taken together to form a compound having theformula:

or a protected derivative thereof, where X is oxygen, nitrogen, orsulfur, and m and n are independently selected integers, such as 1, 2,or 3, and where V, l_(s), and l_(H) are as defined herein. In anotherillustrative aspect of the vitamin receptor binding drug deliveryconjugate intermediate described herein, the drug, or an analog or aderivative thereof, includes an alkylthiol nucleophile.

In another illustrative aspect of the vitamin receptor binding drugdelivery conjugate intermediate described herein, the intermediateincludes compounds having the formulae:

or protected derivatives thereof, where V is the vitamin, or an analogor a derivative thereof, AA is an amino acid, illustratively selectedfrom the naturally occurring amino acids, or stereoisomers thereof, X isnitrogen, oxygen, or sulfur, Y is hydrogen or a substituent,illustratively an electron withdrawing substituent, including but notlimited to nitro, cyano, halo, alkylsulfonyl, a carboxylic acidderivative, and the like, n and m are independently selected integers,such as 1, 2, or 3, and p is an integer such as 1, 2, 3, 4, or 5. AA canalso be any other amino acid, such as any amino acid having the generalformula:—N(R)—(CR′R″)_(q)—C(O)—where R is hydrogen, alkyl, acyl, or a suitable nitrogen protectinggroup, R′ and R″ are hydrogen or a substiuent, each of which isindependently selected in each occurance, and q is an integer such as 1,2, 3, 4, or 5. Illustratively, R′ and/or R″ independently correspond to,but are not limited to, hydrogen or the side chains present on naturallyoccurring amino acids, such as methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, andderivatives and protected derivatives thereof. The above-describedformula includes all stereoisomeric variations. For example, the aminoacid may be selected from asparagine, aspartic acid, cysteine, glutamicacid, lysine, glutamine, arginine, serine, ornitine, threonine, and thelike. In another illustrative aspect of the vitamin receptor bindingdrug delivery conjugate intermediate described herein, the drug, or ananalog or a derivative thereof, includes an alkylthiol nucleophile.

In another embodiment, a process is described for preparing a compoundhaving the formula:

or a protected derivative thereof, where L is a linker comprising(l_(r))_(c), (l_(s))_(a), and (l_(H))_(b), and combinations thereof; andD is a drug, or an analog or a derivative thereof, capable of forming ahydrazone, where (l_(r))_(c), (l_(s))_(a), and (l_(H))_(b), and V are asdefined herein, the process comprising the steps of:

(a) reacting a compound having the formula:

or a protected derivative thereof, with a compound having the formula:

or a protected derivative thereof to form a thiosuccinimide derivative;and

(b) forming a hydrazone derivative of the drug, or an analog or aderivative thereof, with the thiosuccinimide derivative.

In another embodiment, a process is described for preparing a compoundhaving the formula:

where L is a linker comprising (l_(r))_(c), (l_(s))_(a), and(l_(H))_(b), and combinations thereof; and where D is the drug, or ananalog or a derivative thereof, capable of forming a hydrazone, and(l_(r))_(c), (l_(s))_(a), and (l_(H))_(b), and V are as defined herein,the process comprising the step of:

reacting a compound having the -formula:

or a protected derivative thereof, with a compound having the formula:

or a protected derivative thereof.

In another embodiment a pharmaceutical composition is described. Thepharmaceutical composition comprises a drug delivery conjugate inaccordance with the invention, and a pharmaceutically acceptable carriertherefor.

In another embodiment there is described a method for eliminating apopulation of pathogenic cells in a host animal harboring the populationof pathogenic cells wherein the members of the pathogenic cellpopulation have an accessible binding site for a vitamin, or an analogor derivative thereof, and wherein the binding site is uniquelyexpressed, overexpressed, or preferentially expressed by the pathogeniccells. The method comprises the step of administering to the host a drugdelivery conjugate, or a pharmaceutical composition thereof, inaccordance with the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibition of M109 tumor growth by EC112 (Example 9c).

FIG. 2 shows the effect of EC112 (Example 9c) on animal body weights.

FIG. 3 shows the inhibition of M109 tumor growth by EC105 (Example 10a).

FIG. 4 shows the effect of EC105 (Example 10a) on animal body weights.

FIG. 5 shows the lack of inhibition of 4T1 tumor growth by EC105(Example 10a).

FIG. 6 shows the inhibition of M109 tumor growth by EC145 (Example 16b).

FIG. 7 shows the inhibition of M109 tumor growth by EC140 (Example 17a).

FIG. 8 shows the inhibition of L1210 tumor growth by EC136 (Example10b).

FIGS. 9-16 show the inhibition of cellular DNA synthesis by EC135,EC136, EC137, EC138, EC140, EC145, EC158, and EC159 (Examples 17b, 10b,16a, 10c, 17a, 16b, 14e, and 15, respectively).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a vitamin receptor binding drugdelivery conjugate comprising a vitamin receptor binding moiety, abivalent linker (L), and a drug wherein the vitamin receptor bindingmoiety and the drug are each bound to the bivalent linker (L),optionally through an heteroatom linker. The bivalent linker (L)comprises one or more spacer linkers, heteroatom linkers, and releasable(i.e., cleavable) linkers, and combinations thereof, in any order.

The term “releasable linker” as used herein refers to a linker thatincludes at least one bond that can be broken under physiologicalconditions (e.g., a pH-labile, acid-labile, oxidatively-labile, orenzyme-labile bond). It should be appreciated that such physiologicalconditions resulting in bond breaking include standard chemicalhydrolysis reactions that occur, for example, at physiological pH, or asa result of compartmentalization into a cellular organelle such as anendosome having a lower pH than cytosolic pH.

It is understood that a cleavable bond can connect two adjacent atomswithin the releasable linker and/or connect other linkers or V and/or D,as described herein, at either or both ends of the releasable linker. Inthe case where a cleavable bond connects two adjacent atoms within thereleasable linker, following breakage of the bond, the releasable linkeris broken into two or more fragments. Alternatively, in the case where acleavable bond is between the releasable linker and another moiety, suchas an heteroatom linker, a spacer linker, another releasable linker, thedrug, or analog or derivative thereof, or the vitamin, or analog orderivative thereof, following breakage of the bond, the releasablelinker is separated from the other moiety.

The lability of the cleavable bond can be adjusted by, for example,substitutional changes at or near the cleavable bond, such as includingalpha branching adjacent to a cleavable disulfide bond, increasing thehydrophobicity of substituents on silicon in a moiety havingsilicon-oxygen bond that may be hydrolyzed, homologating alkoxy groupsthat form part of a ketal or acetal that may be hydrolyzed, and thelike.

In accordance with the invention, the vitamin receptor binding drugdelivery conjugates can be used to treat disease states characterized bythe presence of a pathogenic cell population in the host wherein themembers of the pathogenic cell population have an accessible bindingsite for a vitamin, or analog or derivative thereof, wherein the bindingsite is uniquely expressed, overexpressed, or preferentially expressedby the pathogenic cells. The selective elimination of the pathogeniccells is mediated by the binding of the vitamin moiety of the vitaminreceptor binding drug delivery conjugate to a vitamin receptor,transporter, or other surface-presented protein that specifically bindsthe vitamin, or analog or derivative thereof, and which is uniquelyexpressed, overexpressed, or preferentially expressed by the pathogeniccells. A surface-presented protein uniquely expressed, overexpressed, orpreferentially expressed by the pathogenic cells is a receptor notpresent or present at lower concentrations on non-pathogenic cellsproviding a means for selective elimination of the pathogenic cells.

For example, surface-expressed vitamin receptors, such as thehigh-affinity folate receptor, are overexpressed on cancer cells.Epithelial cancers of the ovary, mammary gland, colon, lung, nose,throat, and brain have all been reported to express elevated levels ofthe folate receptor. In fact, greater than 90% of all human ovariantumors are known to express large amounts of this receptor. Accordingly,the drug delivery conjugates of the present invention can be used totreat a variety of tumor cell types, as well as other types ofpathogenic cells, such as infectious agents, that preferentially expressvitamin receptors and, thus, have surface accessible binding sites forvitamins, or vitamin analogs or derivatives.

In addition to the vitamins described herein, it is appreciated thatother ligands may be coupled with the drugs and linkers described andcontemplated herein to form ligand-linker-drug conjugates capable offacilitating delivery of the drug to a desired target. These otherligands, in addition to the vitamins and their analogs and derivativesdescribed, may be used to form drug delivery conjugates capable ofbinding to target cells. In general, any ligand of a cell surfacereceptor may be advantageously used as a targeting ligand to which alinker-drug conjugate can be prepared. Illustrative other ligandscontemplated herein include peptide ligands identified from libraryscreens, tumor cell-specific peptides, tumor cell-specific aptamers,tumor cell-specific carbohydrates, tumor cell-specific monoclonal orpolyclonal antibodies, Fab or scFv (i.e., a single chain variableregion) fragments of antibodies such as, for example, an Fab fragment ofan antibody directed to EphA2 or other proteins specifically expressedor uniquely accessible on metastatic cancer cells, small organicmolecules derived from combinatorial libraries, growth factors, such asEGF, FGF, insulin, and insulin-like growth factors, and homologouspolypeptides, somatostatin and its analogs, transferrin, lipoproteincomplexes, bile salts, selecting, steroid hormones, Arg-Gly-Aspcontaining peptides, retinoids, various Galectins, δ-opioid receptorligands, cholecystokinin A receptor ligands, ligands specific forangiotensin AT1 or AT2 receptors, peroxisome proliferator-activatedreceptor λ ligands, β-lactam antibiotics such as penicillin, smallorganic molecules including antimicrobial drugs, and other moleculesthat bind specifically to a receptor preferentially expressed on thesurface of tumor cells or on an infectious organism, antimicrobial andother drugs designed to fit into the binding pocket of a particularreceptor based on the crystal structure of the receptor or other cellsurface protein, ligands of tumor antigens or other moleculespreferentially expressed on the surface of tumor cells,or fragments ofany of these molecules. An example of a tumor-specific antigen thatcould function as a binding site for ligand-immunogen conjugates includeextracellular epitopes of a member of the Ephrin family of proteins,such as EphA2. EphA2 expression is restricted to cell-cell junctions innormal cells, but EpbA2 is distributed over the entire cell surface inmetastatic tumor cells. Thus, EphA2 on metastatic cells would beaccessible for binding to, for example, an Fab fragment of an antibodyconjugated to an immunogen, whereas the protein would not be accessiblefor binding to the Fab fragment on normal cells, resulting in aligand-immunogen conjugate specific for metastatic cancer cells.

The invention further contemplates the use of combinations ofligand-linker-drug conjugates to maximize targeting of the pathogeniccells for elimination.

Illustrative drug delivery conjugates are as follows: V-L-DV-(l_(r))_(c)-D V-(l_(s))_(a)-D V-(l_(s))_(a)-(l_(r))_(c)-DV-(l_(r))_(c)-(l_(s))_(a)-D V-(l_(H))_(b)-(l_(r))_(c)-DV-(l_(r))_(c)-(l_(H))_(b)-D V-(l_(H))_(d)-(l_(r))_(c)-(l_(H))_(e)-DV-(l_(s))_(a)-(l_(H))_(b)-(l_(r))_(c)-DV-(l_(r))_(c)-(l_(H))_(b)-(l_(s))_(a)-DV-(l_(H))_(d)-(l_(s))_(a)-(l_(r))_(c)-(l_(H))_(e)-DV-(l_(H))_(d)-(l_(r))_(c)-(l_(s))_(a)-(l_(H))_(e)-DV-(l_(H))_(d)-(l_(s))_(a)-(l_(H))_(b)-(l_(r))_(c)-(l_(H))_(e)-DV-(l_(H))_(d)-(l_(r))_(c)-(l_(H))_(b)-(l_(s))_(a)-(l_(H))_(e)-DV-(l_(s))_(a)-(l_(r))_(c)-(l_(H))_(b)-DV-[(l_(s))_(a)-(l_(H))_(b)]_(d)-(l_(r))_(c)-(l_(H))_(e)-Dwherein a, b, c, d, and e are each independently 0, 1, 2, 3, or 4,(l_(s)) is a spacer linker, (l_(H)) is an heteroatom linker, and (l_(r))is a releasable linker, V is a vitamin, or analog or derivative thereof,and D is a drug, or analog or derivative thereof, and where the bivalentL encompasses a one or a variety of (l_(s)), (l_(H)), and (l_(r)), inany order and in any combination. It is understood that the foregoingexamples of the bivalent linker L are intended to illustrate, and notlimit, the wide variety of assemblies of l_(H), l_(s), and l_(r) thatthe bivalent linker encompasses.

In one embodiment of the drug delivery conjugate V-L-D, wherein V is thevitamin folic acid; L is not ethylenediamine, according to the formula:

In another embodiment of the drug delivery conjugate V-L-D, wherein V isthe vitamin folic acid, and D is the drug mitomycin C; L is notL-Cys-(S-thioethyl), according to the formula:

L is not L-Asp-L-Arg-L-Asp-L-Cys-(S-thioethyl), according to theformula:

and L is not L-Arg-L-Cys-(S-thioethyl)-L-Ala-L-Gly-OH, according to theformula:

Also contemplated in accordance with the present invention are drugdelivery conjugates where the vitamin, or analog or derivative thereof,is attached to a releasable linker which is attached to the drug througha spacer linker. Furthermore, both the drug and the vitamin, or analogor derivative thereof, can each be attached to spacer linkers, where thespacer linkers are attached to each other through a releasable linker.In addition, both the drug and the vitamin, or analog or derivativethereof, can each be attached to releasable linkers, where thereleasable linkers are attached to each other through a spacer linker.The heteroatom linker may be placed between any two linkers, or betweenany linker and the vitamin, or analog or derivative, or between anylinker and the drug, or analog or derivative. All other possiblepermutations and combinations are also contemplated.

In one embodiment, the present invention provides a vitamin receptorbinding drug delivery conjugate. The drug delivery conjugate consists ofa vitamin receptor binding moiety, bivalent linker (L), and a drug. Thevitamin receptor binding moiety is a vitamin, or an analog or aderivative thereof, capable of binding to vitamin receptors, and thedrug includes analogs or derivatives thereof exhibiting drug activity.The vitamin, or the analog or the derivative thereof, is covalentlyattached to the bivalent linker (L), and the drug, or the analog or thederivative thereof, is also covalently attached to the bivalent linker(L). The bivalent linker (L) comprises one or more spacer linkers,releasable linkers, and heteroatom linkers, and combinations thereof, inany order. For example, the heteroatom linker can be nitrogen, and thereleasable linker and the heteroatom linker can be taken together toform a divalent radical comprising alkyleneaziridin-1-yl,alkylenecarbonylaziridin-1-yl, carbonylalkylaziridin-1-yl,alkylenesulfoxylaziridin-1-yl, sulfoxylalkylaziridin-1-yl,sulfonylalkylaziridin-1-yl, or alkylenesulfonylaziridin-1-yl, whereineach of the releasable linkers is optionally substituted with asubstituent X², as defined below. Alternatively, the heteroatom linkerscan be nitrogen, oxygen, sulfur, and the formulae —(NHR¹NHR²)—, —SO—,—(SO₂)—, and —N(R³)O—, wherein R¹, R², and R³ are each independentlyselected from hydrogen, alkyl, aryl, arylalkyl, substituted aryl,substituted arylalkyl, heteroaryl, substituted heteroaryl, andalkoxyalkyl. In another embodiment, the heteroatom linker can be oxygen,the spacer linker can be 1-alkylenesuccinimid-3-yl, optionallysubstituted with a substituent X¹, as defined below, and the releasablelinkers can be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene,1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, wherein each ofthe releasable linkers is optionally substituted with a substituent X²,as defined below, and wherein the spacer linker and the releasablelinker are each bonded to the heteroatom linker to form asuccinimid-1-ylalkyl acetal or ketal.

The spacer linkers can be carbonyl, thionocarbonyl, alkylene,cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl,1-(carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl,alkylenesulfoxylalkyl, alkylenesulfonylalkyl,carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl,1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of the spacerlinkers is optionally substituted with a substituent X¹, as definedbelow. In this embodiment, the heteroatom linker can be nitrogen, andthe spacer linkers can be alkylenecarbonyl, cycloalkylenecarbonyl,carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, wherein each ofthe spacer linkers is optionally substituted with a substituent X¹, asdefined below, and the spacer linker is bonded to the nitrogen to forman amide. Alternatively, the heteroatom linker can be sulfur, and thespacer linkers can be alkylene and cycloalkylene, wherein each of thespacer linkers is optionally substituted with carboxy, and the spacerlinker is bonded to the sulfur to form a thiol. In another embodiment,the heteroatom linker can be sulfur, and the spacer linkers can be1-alkylenesuccinimid-3-yl and 1-(carbonylalkyl)succinimid-3-yl, and thespacer linker is bonded to the sulfur to form a succinimid-3-ylthiol.

In an alternative to the above-described embodiments, the heteroatomlinker can be nitrogen, and the releasable linker and the heteroatomlinker can be taken together to form a divalent radical comprisingalkyleneaziridin-1-yl, carbonylalkylazindin-1-yl,sulfoxylalkylaziridin-1-yl, or sulfonylalkylaziridin-1-yl, wherein eachof the releasable linkers is optionally substituted with a substituentX², as defined below. In this alternative embodiment, the spacer linkerscan be carbonyl, thionocarbonyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl,1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer linkers isoptionally substituted with a substituent X¹, as defined below, andwherein the spacer linker is bonded to the releasable linker to form anaziridine amide.

The substituents X¹ can be alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl; R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides. In this embodiment the heteroatom linker canbe nitrogen, and the substituent X¹ and the heteroatom linker can betaken together with the spacer linker to which they are bound to form anheterocycle.

The releasable linkers can be methylene, 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,haloalkylenecarbonyl, alkylene(dialkylsilyl), alkylene(alkylarylsilyl),alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl,(diarylsilyl)aryl, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy,oxysulfonylalkyl, iminoalkylidenyl, carbonylalkylideniminyl,iminocycloalkylidenyl, carbonylcycloalkylideniminyl, alkylenethio,alkylenearylthio, and carbonylalkylthio, wherein each of the releasablelinkers is optionally substituted with a substituent X², as definedbelow.

In the preceding embodiment, the heteroatom linker can be oxygen, andthe releasable linkers can be methylene, 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and1-alkoxycycloalkylenecarbonyl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below, and thereleasable linker is bonded to the oxygen to form an acetal or ketal.Alternatively, the heteroatom linker can be oxygen, and the releasablelinker can be methylene, wherein the methylene is substituted with anoptionally-substituted aryl, and the releasable linker is bonded to theoxygen to form an acetal or ketal. Further, the heteroatom linker can beoxygen, and the releasable linker can be sulfonylalkyl, and thereleasable linker is bonded to the oxygen to form an alkylsulfonate.

In another embodiment of the above releasable linker embodiment, theheteroatom linker can be nitrogen, and the releasable linkers can beiminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl, andcarbonylcycloalkylideniminyl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below, and thereleasable linker is bonded to the nitrogen to form an hydrazone. In analternate configuration,-the hydrazone may be acylated with a carboxylicacid derivative, an orthoformate derivative, or a carbamoyl derivativeto form various acylhydrazone releasable linkers.

Alternatively, the heteroatom linker can be oxygen, and the releasablelinkers can be alkylene(dialkylsilyl), alkylene(alkylarylsilyl),alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, and(diarylsilyl)aryl, wherein each of the releasable linkers is optionallysubstituted with a substituent X², as defined below, and the releasablelinker is bonded to the oxygen to form a silanol.

In the above releasable linker embodiment, the drug can include anitrogen atom, the heteroatom linker can be nitrogen, and the releasablelinkers can be carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl,carbonyl(biscarboxyaryl)carbonyl, and the releasable linker can bebonded to the heteroatom nitrogen to form an amide, and also bonded tothe drug nitrogen to form an amide.

In the above releasable linker embodiment, the drug can include anoxygen atom, the heteroatom linker can be nitrogen, and the releasablelinkers can be carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl,carbonyl(biscarboxyaryl)carbonyl, and the releasable linker can bebonded to the heteroatom linker nitrogen to form an amide, and alsobonded to the drug oxygen to form an ester.

The substituents X² can be alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides. In this embodiment the heteroatom linker canbe nitrogen, and the substituent X² and the heteroatom linker can betaken together with the releasable linker to which they are bound toform an heterocycle.

The heterocycles can be pyrrolidines, piperidines, oxazolidines,isoxazolidines, thiazolidines, isothiazolidines, pyrrolidinones,piperidinones, oxazolidinones, isoxazolidinones, thiazolidinones,isothiazolidinones, and succinimides.

The drug can be a mitomycin, a mitomycin derivative, or a mitomycinanalog, and, in this embodiment, the releasable linkers can becarbonylalkylthio, carbonyltetrahydro-2H-pyranyl,carbonyltetrahydrofuranyl,1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of thereleasable linkers is optionally substituted with a substituent X², andwherein the aziridine of the mitomycin is bonded to the releasablelinker to form an acylaziridine.

The drug can include a nitrogen atom, and the releasable linker can behaloalkylenecarbonyl, optionally substituted with a substituent X², andthe releasable linker is bonded to the drug nitrogen to form an amide.

The drug can include an oxygen atom, and the releasable linker can behaloalkylenecarbonyl, optionally substituted with a substituent X², andthe releasable linker is bonded to the drug oxygen to form an ester.

The drug can include a double-bonded nitrogen atom, and in thisembodiment, the releasable linkers can be alkylenecarbonylamino and1-(alkylenecarbonylamino)succinimid-3-yl, and the releasable linker canbe bonded to the drug nitrogen to form an hydrazone.

The drug can include a sulfur atom, and in this embodiment, thereleasable linkers can be alkylenethio and carbonylalkylthio, and thereleasable linker can be bonded to the drug sulfur to form a disulfide.

The vitamin can be folate which includes a nitrogen, and in thisembodiment, the spacer linkers can be alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl,1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer linkers isoptionally substituted with a substituent X¹, and the spacer linker isbonded to the folate nitrogen to form an imide or an alkylamide. In thisembodiment, the substituents X¹ can be alkyl, hydroxyalkyl, amino,aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, sulfhydrylalkyl,alkylthioalkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, carboxy, carboxyalkyl, guanidinoalkyl, R⁴-carbonyl,R⁵-carbonylalkyl, R⁶-acylamino, and R⁷-acylaminoalkyl, wherein R⁴ and R⁵are each independently selected from amino acids, amino acidderivatives, and peptides, and wherein R⁶ and R⁷ are each independentlyselected from amino acids, amino acid derivatives, and peptides.

The term “alkyl” as used herein refers to a monovalent linear chain ofcarbon atoms that may be optionally branched, such as methyl, ethyl,propyl, 3-methylpentyl, and the like.

The term “cycloalkyl” as used herein refers to a monovalent chain ofcarbon atoms, a portion of which forms a ring, such as cyclopropyl,cyclohexyl, 3-ethylcyclopentyl, and the like.

The term “alkylene” as used herein refers to a bivalent linear chain ofcarbon atoms that may be optionally branched, such as methylene,ethylene, propylene, 3-methylpentylene, and the like.

The term “cycloalkylene” as used herein refers to a bivalent chain ofcarbon atoms, a portion of which forms a ring, such ascycloprop-1,1-diyl, cycloprop-1,2-diyl, cyclohex-1,4-diyl,3-ethylcyclopent-1,2-diyl, 1-methylenecyclohex-4-yl, and the like.

The term “heterocycle” as used herein refers to a monovalent chain ofcarbon and heteroatoms, wherein the heteroatoms are selected fromnitrogen, oxygen, and sulfur, a portion of which, including at least oneheteroatom, form a ring, such as aziridine, pyrrolidine, oxazolidine,3-methoxypyrrolidine, 3-methylpiperazine, and the like.

The term “alkoxy” as used herein refers to alkyl as defined hereincombined with a terminal oxygen, such as methoxy, ethoxy, propoxy,3-methylpentoxy, and the like.

The term “halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

The term “aryl” as used herein refers to an aromatic mono or polycyclicring of carbon atoms, such as phenyl, naphthyl, and the like.

The term “heteroaryl” as used herein refers to an aromatic mono orpolycyclic ring of carbon atoms and at least one heteroatom selectedfrom nitrogen, oxygen, and sulfur, such as pyridinyl, pyrimidinyl,indolyl, benzoxazolyl, and the like.

The term “substituted aryl” or “substituted heteroaryl” as used hereinrefers to aryl or heteroaryl substituted with one or more substituentsselected, such as halo, hydroxy, amino, alkyl or dialkylamino, alkoxy,alkylsulfonyl, cyano, nitro, and the like.

The term “iminoalkylidenyl” as used herein refers to a divalent radicalcontaining alkylene as defined herein and a nitrogen atom, where theterminal carbon of the alkylene is double-bonded to the nitrogen atom,such as the formulae —(CH)═N—, —(CH₂)₂(CH)═N—, —CH₂C(Me)═N—, and thelike.

The term “amino acid” as used herein refers generally toaminoalkylcarboxylate, where the alkyl radical is optionally substitutedwith alkyl, hydroxy alkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl,and the like, including groups corresponding to the naturally occurringamino acids, such as serine, cysteine, methionine, aspartic acid,glutamic acid, and the like.

The term “arylalkyl” refers to aryl as defined herein substituted withan alkylene group, as defined herein, such as benzyl, phenethyl,α-methylbenzyl, and the like.

It should be understood that the above-described terms can be combinedto generate chemically-relevant groups, such as “alkoxyalkyl” referringto methyloxymethyl, ethyloxyethyl, and the like, and “haloalkoxyalkyl”referring to trifluoromethyloxyethyl,1,2-difluoro-2-chloroeth-1-yloxypropyl, and the like.

The term “amino acid derivative” as used herein refers generally toaminoalkylcarboxylate, where the amino radical or the carboxylateradical are each optionally substituted with alkyl, carboxylalkyl,alkylamino, and the like, or optionally protected; and the interveningdivalent alkyl fragment is optionally substituted with alkyl, hydroxyalkyl, sulfhydrylalkyl, aminoalkyl, carboxyalkyl, and the like,including groups corresponding to the side chains found in naturallyoccurring amino acids, such as are found in serine, cysteine,methionine, aspartic acid, glutamic acid, and the like.

The term “peptide” as used herein refers generally to a series of aminoacids and amino acid analogs and derivatives covalently linked one tothe other by amide bonds.

The releasable linker includes at least one bond that can be broken orcleaved under physiological conditions (e.g., a pH-labile, acid-labile,oxidatively-labile, or enzyme-labile bond). The cleavable bond or bondsmay be present in the interior of a cleavable linker and/or at one orboth ends of a cleavable linker. It is appreciated that the lability ofthe cleavable bond may be adjusted by including functional groups orfragments within the bivalent linker L that are able to assist orfacilitate such bond breakage, also termed anchimeric assistance. Inaddition, it is appreciated that additional functional groups orfragments may be included within the bivalent linker L that are able toassist or facilitate additional fragmentation of the vitamin receptorbinding drug conjugates after bond breaking of the releasable linker.

Illustrative mechanisms for bond cleavage of the releasable linkerinclude oxonium-assisted cleavage as follows:

where Z is the vitamin, or analog or derivative thereof, or the drug, oranalog or derivative thereof, or each is a vitamin or drug moiety inconjunction with other portions of the bivalent linker, such as a drugor vitamin moiety including one or more spacer linkers, heteroatomlinkers, and/or other releasable linkers. In this embodiment,acid-catalyzed elimination of the carbamate leads to the release of CO₂and the nitrogen-containing moiety attached to Z, and the formation of abenzyl cation, which may be trapped by water, or any other Lewis base.

Another illustrative mechanism for cleavage of bonds connected to orcontained within releasable linkers, which may form part of the bivalentlinker L, include the following beta-elimination and vinylogousbeta-elimination mechanisms:

where X is a nucleophile, GSH, glutathione, or bioreducing agent, andthe like, and either of Z or Z′ is the vitamin, or analog or derivativethereof, or the drug, or analog or derivative thereof, or a vitamin ordrug moiety in conjunction with other portions of the bivalent linker.It is appreciated that the bond cleavage may also occur by acidcatalyzed elimination of the carbamate moiety, which may beanchimerically assisted by the stabilization provided by either the arylgroup of the beta sulfur or disulfide illustrated in the above examples.In those variations of this embodiment, the releasable linker is thecarbamate moiety.

Another illustrative mechanism involves an arrangement of thereleasable, spacer, and heteroatom linkers in such a way that subsequentto the cleavage of a bond in the bivalent linker, released functionalgroups chemically assist the breakage or cleavage of additional bonds,also termed anchimeric assisted cleavage or breakage. An illustrativeembodiment of such a bivalent linker or portion thereof includescompounds having the formula:

where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is aninteger selected from 0, 1, 2, and 3, R is hydrogen, or a substituent,including a substituent capable of stabilizing a positive chargeinductively or by resonance on the aryl ring, such as alkoxy, and thelike, and either of Z or Z′ is the vitamin, or analog or derivativethereof, or the drug, or analog or derivative thereof, or a vitamin ordrug moiety in conjunction with other portions of the bivalent linker.It is appreciated that other substituents may be present on the arylring, the benzyl carbon, the carbamate nitrogen, the alkanoic acid, orthe methylene bridge, including but not limited to hydroxy, alkyl,alkoxy, alkylthio, halo, and the like. Assisted cleavage may includemechanisms involving benzylium intermediates, benzyne intermediates,lactone cyclization, oxonium intermediates, beta-elimination, and thelike. It is further appreciated that, in addition to fragmentationsubsequent to cleavage of the releasable linker, the initial cleavage ofthe releasable linker may be facilitated by an anchimerically assistedmechanism.

In this embodiment, the hydroxyalkanoic acid, which may cyclize,facilitates cleavage of the methylene bridge, by for example an oxoniumion, and facilitates bond cleavage or subsequent fragmentation afterbond cleavage of the releasable linker. Alternatively, acid catalyzedoxonium ion-assisted cleavage of the methylene bridge may begin acascade of fragmentation of this illustrative bivalent linker, orfragment thereof. Alternatively, acid-catalyzed hydrolysis of thecarbamate may facilitate the beta elimination of the hydroxyalkanoicacid, which may cyclize, and facilitate cleavage of methylene bridge, byfor example an oxonium ion. It is appreciated that other chemicalmechanisms of bond breakage or cleavage under the metabolic,physiological, or cellular conditions described herein may initiate sucha cascade of fragmentation. It is appreciated that other chemicalmechanisms of bond breakage or cleavage under the metabolic,physiological, or cellular conditions described herein may initiate sucha cascade of fragmentation.

The drug delivery conjugates described herein can be prepared byart-recognized synthetic methods. The synthetic methods are chosendepending upon the selection of the heteroatom linkers, and thefunctional groups present on the spacer linkers and the releasablelinkers. In general, the relevant bond forming reactions are describedin Richard C. Larock, “Comprehensive Organic Transformations, a guide tofunctional group preparations,” VCH Publishers, Inc. New York (1989),and in Theodora E. Greene & Peter G. M. Wuts, “Protective Groups ionOrganic Synthesis,” 2d edition, John Wiley & Sons, Inc. New York (1991),the disclosures of which are incorporated herein by reference.

General Amide and Ester Formation.

For example, where the heteroatom linker is a nitrogen atom, and theterminal functional group present on the spacer linker or the releasablelinker is a carbonyl group, the required amide group can be obtained bycoupling reactions or acylation reactions of the correspondingcarboxylic acid or derivative, where L is a suitably-selected leavinggroup such as halo, triflate, pentafluorophenoxy, trimethylsilyloxy,succinimide-N-oxy, and the like, and an amine, as illustrated in Scheme1.

Coupling reagents include DCC, EDC, RRDQ, CGI, HBTU, TBTU, HOBT/DCC,HOBT/EDC, BOP-Cl, PyBOP, PyBroP, and the like. Alternatively, the parentacid can be converted into an activated carbonyl derivative, such as anacid chloride, a N-hydroxysuccinimidyl ester, a pentafluorophenyl ester,and the like. The amide-forming reaction can also be conducted in thepresence of a base, such as triethylamine, diisopropylethylamine,N,N-dimethyl-4-aminopyridine, and the like. Suitable solvents forforming amides described herein include CH₂Cl₂, CHCl₃, THF, DMF, DMSO,acetonitrile, EtOAc, and the like. Illustratively, the amides can beprepared at temperatures in the range from about −15° C. to about 80°C., or from about 0° C. to about 45° C. Amides can be formed from, forexample, nitrogen-containing aziridine rings, carbohydrates, andα-halogenated carboxylic acids. Illustrative carboxylic acid derivativesuseful for forming amides include compounds having the formulae:

and the like, where n is an integer such as 1, 2, 3, or 4.

Similarly, where the heteroatom linker is an oxygen atom and theterminal functional group present on the spacer linker or the releasablelinker is a carbonyl group, the required ester group can be obtained bycoupling reactions of the corresponding carboxylic acid or derivative,and an alcohol.

Coupling reagents include DCC, EDC, CDI, BOP, PyBOP, isopropenylchloroformate, EEDQ, DEAD, PPh₃, and the like. Solvents include CH₂Cl₂,CHCl₃, TBF, DMF, DMSO, acetonitrile, EtOAc, and the like. Bases includetriethylamine, diisopropyl-ethylamine, and N,N-dimethyl-4-aminopyridine.Alternatively, the parent acid can be converted into an activatedcarbonyl derivative, such as an acid chloride, a N-hydroxysuccinimidylester, a pentafluorophenyl ester, and the like.

General Ketal and Acetal Formation.

Furthermore, where the heteroatom linker is an oxygen atom, and thefunctional group present on the spacer linker or the releasable linkeris 1-alkoxyalkyl, the required acetal or ketal group can be formed byketal and acetal forming reactions of the corresponding alcohol and anenol ether, as illustrated in Scheme 2.

Solvents include alcohols, CH₂Cl₂, CHCl₃, THF, diethylether, DMF, DMSO,acetonitrile, EtOAc, and the like. The formation of such acetals andketals can be accomplished with an acid catalyst. Where the heteroatomlinker comprises two oxygen atoms, and the releasable linker ismethylene, optionally substituted with a group X² as described herein,the required symmetrical acetal or ketal group can be illustrativelyformed by acetal and ketal forming -reactions from the correspondingalcohols and an aldehyde or ketone, as illustrated in Scheme 3.

Alternatively, where the methylene is substituted with anoptionally-substituted aryl group, the required acetal or ketal may beprepared stepwise, where L is a suitably selected leaving group such ashalo, trifluoroacetoxy, triflate, and the like, as illustrated in Scheme4. The process illustrated in Scheme 4 is a conventional preparation,and generally follows the procedure reviewed by R. R. Schmidt et al.,Chem. Rev., 2000, 100, 4423-42, the disclosure of which is incorporatedherein by reference.

The resulting arylalkyl ether is treated with an oxidizing agent, suchas DDQ, and the like, to generate an intermediate oxonium ion that issubsequently treated with another alcohol to generate the acetal orketal.

General Succinimide Formation.

Furthermore, where the heteroatom linker is, for example, a nitrogen,oxygen, or sulfur atom, and the functional group present on the spacerlinker or the releasable linker is a succinimide derivative, theresulting carbon-heteroatom bond can be formed by a Michael addition ofthe corresponding amine, alcohol, or thiol, and a maleimide derivative,where X is the heteroatom linker, as illustrated in Scheme 5.

Solvents for performing the Michael addition include THF, EtOAc, CH₂Cl₂,DMF, DMSO, H₂O and the like. The formation of such Michael adducts canbe accomplished with the addition of equimolar amounts of bases, such astriethylamine, Hunig's base or by adjusting the pH of water solutions to6.0-7.4. It is appreciated that when the heteroatom linker is an oxygenor nitrogen atom, reaction conditions may be adjusted to facilitate theMichael addition, such as, for example, by using higher reactiontemperatures, adding catalysts, using more polar solvents, such as DMF,DMSO, and the like, and activating the maleimide with silylatingreagents.

General Silyloxy Formation.

Furthermore, where the heteroatom linker is an oxygen atom, and thefunctional group present on the spacer linker or the releasable linkeris a silyl derivative, the required silyloxy group may be formed byreacting the corresponding silyl derivative, and an alcohol, where L isa suitably selected leaving group such as halo, trifluoroacetoxy,triflate, and the like, as illustrated in Scheme 6.

Silyl derivatives include properly functionalized silyl derivatives suchas vinylsulfonoalkyl diaryl, or diaryl, or alkyl aryl silyl chloride.Instead of a vinylsulfonoalkyl group, a β-chloroethylsulfonoalkylprecursor may be used. Any aprotic and anhydrous solvent and anynitrogen-containing base may serve as a reaction medium. The temperaturerange employed in this transformation may vary between −78° C. and 80°C.

General Hydrazone Formation.

Furthermore, where the heteroatom linker is a nitrogen atom, and thefunctional group present on the spacer linker or the releasable linkeris an iminyl derivative, the required hydrazone group can be formed byreacting the corresponding aldehyde or ketone, and a hydrazine oracylhydrazine derivative, as illustrated in Scheme 7, equations (1) and(2) respectively.

Solvents that can be used include THF, EtOAc, CH₂Cl₂, CHCl₃, CCl₄, DMF,DMSO, MeOH and the like. The temperature range employed in thistransformation may vary between 0° C. and 80° C. Any acidic catalystsuch as a mineral acid, H₃CCOOH, F₃CCOOH, p-TsOH.H₂O, pyridiniump-toluene sulfonate, and the like can be used. In the case of theacylhydrazone in equation (2), the acylhydrazone may be prepared byinitially acylating hydrazine with a suitable carboxylic acid orderivative, as generally described above in Scheme 1, and subsequentlyreacting the acylhydrazide with the corresponding aldehyde or ketone toform the acylhydrazone. Alternatively, the hydrazone functionality maybe initially formed by reacting hydrazine with the correspondingaldehyde or ketone. The resulting hydrazone may subsequently be acylatedwith a suitable carboxylic acid or derivative, as generally describedabove in Scheme 1.

General Disulfide Formation.

Furthermore, where the heteroatom linker is a sulfur atom, and thefunctional group present on the releasable linker is an alkylenethiolderivative, the required disulfide group can be formed by reacting thecorresponding alkyl or aryl sulfonylthioalkyl derivative, or thecorresponding heteroaryldithioalkyl derivative such as apyridin-2-yldithioalkyl derivative, and the like, with an alkylenethiolderivative, as illustrated in Scheme 8.

Solvents that can be used are THF, EtOAc, CH₂Cl₂, CHCl₃, CCl₄, DMF,DMSO, and the like. The temperature range employed in thistransformation may vary between 0° C. and 80° C. The required alkyl oraryl sulfonylthioalkyl derivative may be prepared using art-recognizedprotocols, and also according to the method of Ranasinghe and Fuchs,Synth. Commun. 18(3), 227-32 (1988), the disclosure of which isincorporated herein by reference. Other methods of preparingunsymmetrical dialkyl disulfides are based on a transthiolation ofunsymmetrical heteroaryl-alkyl disulfides, such as 2-thiopyridinyl,3-nitro-2-thiopyridinyl, and like disulfides, with alkyl thiol, asdescribed in WO 88/01622, European Patent Application No. 0116208A1, andU.S. Pat. No. 4,691,024, the disclosures of which are incorporatedherein by reference.

General Carbonate Formation.

Furthermore, where the heteroatom linker is an oxygen atom, and thefunctional group present on the spacer linker or the releasable linkeris an alkoxycarbonyl derivative, the required carbonate group can beformed by reacting the corresponding hydroxy-substituted compound withan activated alkoxycarbonyl derivative where L is a suitable leavinggroup, as illustrated in Scheme 9.

Solvents that can be used are THF, EtOAc, CH₂Cl₂, CHCl₃, CCl₄, DMF,DMSO, and the like. The temperature range employed in thistransformation may vary between 0° C. and 80° C. Any basic catalyst suchas an inorganic base, an amine base, a polymer bound base, and the likecan be used to facilitate the reaction.

General Semicarbazone Formation.

Furthermore, where the heteroatom linker is a nitrogen atom, and thefunctional group present on one spacer linker or the releasable linkeris an iminyl derivative, and the functional group present on the otherspacer linker or the other releasable linker is an alkylamino orarylaminocarbonyl derivative, the required semicarbazone group can beformed by reacting the corresponding aldehyde or ketone, and asemicarbazide derivative, as illustrated in Scheme 10.

Solvents that can be used are THF, EtOAc, CH₂Cl₂, CHCl₃, CCl₄, DMF,DMSO, MeOH and the like. The temperature range employed in thistransformation may vary between 0° C. and 80° C. Any acidic catalystsuch as a mineral acid, H₃CCOOH, F₃CCOOH, p-TsOH.H₂O, pyridiniump-toluene sulfonate, and the like can be used. In addition, in formingthe semicarbazone, the hydrazone functionality may be initially formedby reacting hydrazine with the corresponding aldehyde or ketone. Theresulting hydrazone may subsequently by acylated with an isocyanate or acarbamoyl derivative, such as a carbamoyl halide, to form thesemicarbazone. Alternatively, the corresponding semicarbazide may beformed by reacting hydrazine with an isocyanate or carbamoyl derivative,such as a carbamoyl halide to form a semicarbazide. Subsequently, thesemicarbazide may be reacted with the corresponding aldehyde or ketoneto form the semicarbazone.

General Sulfonate Formation.

Furthermore, where the heteroatom linker is an oxygen atom, and thefunctional group present on the spacer linker or the releasable linkeris sulfonyl derivative, the required sulfonate group can be-formed byreacting the corresponding hydroxy-substituted compound with anactivated sulfonyl derivative where L is a suitable leaving group suchas halo, and the like, as illustrated in Scheme 11.

Solvents that can be used are THF, EtOAc, CH₂Cl₂, CHCl₃, CCl₄, and thelike. The temperature range employed in this transformation may varybetween 0° C. and 80° C. Any basic catalyst such as an inorganic base,an amine base, a polymer bound base, and the like can be used tofacilitate the reaction.

General Formation of Folate-Peptides.

The folate-containing peptidyl fragment Pte-Glu-(AA)_(n)-NH(CHR₂)CO₂H(3) is prepared by a polymer-supported sequential approach usingstandard methods, such as the Fmoc-strategy on an acid-sensitiveFmoc-AA-Wang resin (1), as shown in Scheme 12.

In this illustrative embodiment of the processes described herein, R₁ isFmoc, R₂ is the desired appropriately-protected amino acid side chain,and DIPEA is diisopropylethylamine. Standard coupling procedures, suchas PyBOP and others described herein or known in the art are used, wherethe coupling agent is illustratively applied as the activating reagentto ensure efficient coupling. Fmoc protecting groups are removed aftereach coupling step under standard conditions, such as upon treatmentwith piperidine, tetrabutylammonium fluoride (TBAF), and the like.Appropriately protected amino acid building blocks, such asFmoc-Glu-OtBu, N¹⁰-TFA-Pte-OH, and the like, are used, as described inScheme 12, and represented in step (b) by Fmoc-AA-OH. Thus, AA refers toany amino acid starting material, that is appropriatedly protected. Itis to be understood that the term amino acid as used herein is intendedto refer to any reagent having both an amine and a carboxylic acidfunctional group separated by one or more carbons, and includes thenaturally occurring alpha and beta amino acids, as well as amino acidderivatives and analogs of these amino acids. In particular, amino acidshaving side chains that are protected, such as protected serine,threonine, cysteine, aspartate, and the like may also be used in thefolate-peptide synthesis described herein. Further, gamma, delta, orlonger homologous amino acids may also be included as starting materialsin the folate-peptide synthesis described herein. Further, amino acidanalogs having homologous side chains, or alternate branchingstructures, such as norleucine, isovaline, β-methyl threonine, β-methylcysteine, β,β-dimethyl cysteine, and the like, may also be included asstarting materials in the folate-peptide synthesis described herein.

The coupling sequence (steps (a) & (b)) involving Fmoc-AA-OH isperformed “n” times to prepare solid-support peptide 2, where n is aninteger and may equal 0 to about 100. Following the last coupling step,the remaining Fmoc group is removed (step (a)), and the peptide issequentially coupled to a glutamate derivative (step (c)), deprotected,and coupled to TFA-protected pteroic acid (step (d)). Subsequently, thepeptide is cleaved from the polymeric support upon treatment withtrifluoroacetic acid, ethanedithiol, and triisopropylsilane (step (e)).These reaction conditions result in the simultaneous removal of thet-Bu, t-Boc, and Trt protecting groups that may form part of theappropriately-protected amino acid side chain. The TFA protecting groupis removed upon treatment with base (step (f)) to provide thefolate-containing peptidyl fragment 3.

The spacer and releasable linkers, and the heteroatom linkers can becombined in a variety of ways. Illustratively, the linkers are attachedto each other through an heteroatom linker, such asalkylene--amino--alkylenecarbonyl,alkylene--thio--carbonylalkylsuccinimid-3-yl, and the like, as furtherillustrated by the following formulae, where the integers x and y are 1,2, 3, 4, or 5:

Another illustrative embodiment of the linkers described herein, includereleasable linkers that cleave under the conditions described herein bya chemical mechanism involving beta elimination. In one aspect, suchreleasable linkers include beta-thio, beta-hydroxy, and beta-aminosubstituted carboxylic acids and derivatives thereof, such as esters,amides, carbonates, carbamates, and ureas. In another aspect, suchreleasable linkers include 2- and 4-thioarylesters, carbamates, andcarbonates.

Furthermore, attachment of the vitamin or drug to the heteroatom linkercan be made through a reactive functional group present on the drug orvitamin that has been converted to an heteroatom linker, such asconversion of the aclamycin ketone to the corresponding hydrazone,conversion of folic acid to the corresponding amide, and the like, asillustrated by the following formulae:

The bivalent linker (L) comprises-one or more components selected fromspacer linkers, releasable linkers, heteroatom linkers, and combinationsthereof in any order. For example, the spacer linkers, releasablelinkers, and heteroatom linkers, and combinations thereof, illustratedin Tables 1 and 2 are contemplated. These lists of linkers are notcomprehensive, but are merely illustrative and should not be interpretedas limiting the invention described herein. The asterisks present on theforegoing structures, as well as those shown in Tables 1 and 2, identifyillustrative points of attachment for additional spacer, releasable, orheteroatom linkers, or for the drug or the vitamin component of thevitamin receptor binding drug delivery conjugate. It is understood thatthe bivalent linker L comprises one or more spacer linkers, releasablelinkers, and heteroatom linkers, including those illustrated in Tables 1and 2, and such spacer linkers, releasable linkers, and heteroatomlinkers may be combined in any order to form the bivalent linker L.TABLE 1 Contemplated linkers, and combinations of certain spacer andheteroatom linkers.

TABLE 2 Contemplated linkers, and combinations of certain releasable andheteroatom linkers.

The drug delivery conjugates of the invention may also be made fromintermediates. In one embodiment, a compound of the formula:V-L-Z¹can be made, where Z¹ is an electrophile, nucleophile, or precursor,suitable for facilitating attachment of the drug, or analog orderivative thereof.

In one aspect, Z¹ can be a leaving group that allows attachment of thedrug through a nucleophilic residue present on the drug, or analog orderivative thereof, such as an heteroatom, for example, nitrogen.

In another aspect, Z¹ can be a nucleophile, such as an heteroatom, forexample nitrogen, capable of displacing a leaving group present on thedrug, or analog or derivative thereof, such as a carboxylic acidderivative, for example, an acid chloride.

In another aspect, Z¹ can be a precursor, such as a nitro group capableof being elaborated into a nucleophilic nitrogen via a reductionreaction, or an ester capable of being elaborated into an electrophilicacid chloride by sequential hydrolysis and chlorination. It should beappreciated that Z¹ can be an heteroatom linker.

In another embodiment, the drug delivery conjugates can be made fromintermediates as follows:Z²-L-Dwhere Z² is an electrophile, nucleophile, or precursor, suitable forfacilitating attachment of the vitamin, or analog or derivative thereof.

In one aspect, Z¹ can be a leaving group that allows attachment of thevitamin through a nucleophilic residue present on the vitamin, or analogor derivative thereof, such as an heteroatom, for example, nitrogen.

In another aspect, Z¹ can be a nucleophile, such as an heteroatom, forexample, nitrogen, capable of displacing a leaving group present on thevitamin, or analog or derivative thereof, such as a carboxylic acidderivative, for example, an acid chloride.

In another aspect, Z¹ can be a precursor, such as a nitro group capableof being elaborated into a nucleophilic nitrogen via a reductionreaction, or an ester capable of being elaborated into an electrophilicacid chloride by sequential hydrolysis and chlorination. It should beappreciated that Z² can be an heteroatom linker.

In another embodiment, the bivalent linker (L) may be separatelysynthesized, then attached to the vitamin and the drug in subsequentsteps, such as by preparing an intermediate a compound of formula:Z¹-L-Z²where Z¹ and Z² are each independently selected, and are as definedabove.

The drug delivery conjugates of the present invention can be used forboth human clinical medicine and veterinary applications. Thus, the hostanimal harboring the population of pathogenic cells and treated with thevitamin receptor binding drug delivery conjugates can be human or, inthe case of veterinary applications, can be a laboratory, agricultural,domestic, or wild animal. The present invention can be applied to hostanimals including, but not limited to, humans, laboratory animals suchrodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys,chimpanzees, domestic animals such as dogs, cats, and rabbits,agricultural animals such as cows, horses, pigs, sheep, goats, and wildanimals in captivity such as bears, pandas, lions, tigers, leopards,elephants, zebras, giraffes, gorillas, dolphins, and whales.

The invention is applicable to populations of pathogenic cells thatcause a variety of pathologies in these host animals. In accordance withthe invention “pathogenic cells” means cancer cells, infectious agentssuch as bacteria and viruses, bacteria- or virus-infected cells,activated macrophages capable of causing a disease state, and any othertype of pathogenic cells that uniquely express, preferentially express,or overexpress vitamin receptors or receptors that bind analogs orderivatives of vitamins. Pathogenic cells can also include any cellscausing a disease state for which treatment with the vitamin receptorbinding drug delivery conjugates of the present invention results inreduction of the symptoms of the disease. For example, the pathogeniccells can be host cells that are pathogenic under some circumstancessuch as cells of the immune system that are responsible for graft versushost disease, but not pathogenic under other circumstances.

Thus, the population of pathogenic cells can be a cancer cell populationthat is tumorigenic, including benign tumors and malignant tumors, or itcan be non-tumorigenic. The cancer cell population can arisespontaneously or by such processes as mutations present in the germlineof the host animal or somatic mutations, or it can be chemically-,virally-, or radiation-induced. The invention can be utilized to treatsuch cancers as carcinomas, sarcomas, lymphomas, Hodgekin's disease,melanomas, mesotheliomas, Burkitt's lymphoma, nasopharyngeal carcinomas,leukemias, and myelomas. The cancer cell population can include, but isnot limited to, oral, thyroid, endocrine, skin, gastric, esophageal,laryngeal, pancreatic, colon, bladder, bone, ovarian, cervical, uterine,breast, testicular, prostate, rectal, kidney, liver, and lung cancers.

In embodiments where the pathogenic cell population is a cancer cellpopulation, the effect of conjugate administration is a therapeuticresponse measured by reduction or elimination of tumor mass or ofinhibition of tumor cell proliferation. In the case of a tumor, theelimination can be an elimination of cells of the primary tumor or ofcells that have metastasized or are in the process of dissociating fromthe primary tumor. A prophylactic treatment with the vitamin receptorbinding drug delivery conjugate to prevent return of a tumor after itsremoval by any therapeutic approach including surgical removal of thetumor, radiation therapy, chemotherapy, or biological therapy is alsocontemplated in accordance with this invention. The prophylactictreatment can be an initial treatment with the drug delivery conjugate,such as treatment in a multiple dose daily regimen, and/or can be anadditional treatment or series of treatments after an interval of daysor months following the initial treatment (s). Accordingly, eliminationof any of the pathogenic cell populations treated in accordance withthis invention includes reduction in the number of pathogenic cells,inhibition of proliferation of pathogenic cells, a prophylactictreatment that prevents return of pathogenic cells, or a treatment ofpathogenic cells that results in reduction of the symptoms of disease.

In cases where cancer cells are being eliminated, the method of thepresent invention can be used in combination with surgical removal of atumor, radiation therapy, chemotherapy, or biological therapies such asother immunotherapies including, but not limited to, monoclonal antibodytherapy, treatment with immunomodulatory agents, adoptive transfer ofimmune effector cells, treatment with hematopoietic growth factors,cytokines and vaccination.

The invention is also applicable to populations of pathogenic cells thatcause a variety of infectious diseases. For example, the presentinvention is applicable to such populations of pathogenic cells asbacteria, fungi, including yeasts, viruses, virus-infected cells,mycoplasma, and parasites. Infectious organisms that can be treated withthe drug delivery conjugates of the present invention are anyart-recognized infectious organisms that cause pathogenesis in ananimal, including such organisms as bacteria that are gram-negative orgram-positive cocci or bacilli. For example, Proteus species, Klebsiellaspecies, Providencia species, Yersinia species, Erwinia species,Enterobacter species, Salmonella species, Serratia species, Aerobacterspecies, Escherichia species, Pseudomonas species, Shigella species,Vibrio species, Aeromonas species, Campylobacter species, Streptococcusspecies, Staphylococcus species, Lactobacillus species, Micrococcusspecies, Moraxella species, Bacillus species, Clostridium species,Corynebacterium species, Eberthella species, Micrococcus species,Mycobacterium species, Neisseria species, Haemophilus species,Bacteroides species, Listeria species, Erysipelothrix species,Acinetobacter species, Brucella species, Pasteurella species, Vibriospecies, Flavobacterium species, Fusobacterium species, Streptobacillusspecies, Calymmatobacterium species, Legionella species, Treponemaspecies, Borrelia species, Leptospira species, Actinomyces species,Nocardia species, Rickettsia species, and any other bacterial speciesthat causes disease in a host can be treated with the drug deliveryconjugates of the invention.

Of particular interest are bacteria that are resistant to antibioticssuch as antibiotic-resistant Streptococcus species and Staphlococcusspecies, or bacteria that are susceptible to antibiotics, but causerecurrent infections treated with antibiotics so that resistantorganisms eventually develop. Bacteria that are susceptible toantibiotics, but cause recurrent infections treated with antibiotics sothat resistant organisms eventually develop, can be treated with thedrug delivery conjugates of the present invention in the absence ofantibiotics, or in combination with lower doses of antibiotics thanwould normally be administered to a patient, to avoid the development ofthese antibiotic-resistant bacterial strains.

Viruses, such as DNA and RNA viruses, can also be treated in accordancewith the invention. Such viruses include, but are not limited to, DNAviruses such as papilloma viruses, parvoviruses, adenoviruses,herpesviruses and vaccinia viruses, and RNA viruses, such asarenaviruses, coronaviruses, rhinoviruses, respiratory syncytialviruses, influenza viruses, picornaviruses, paramyxoviruses, reoviruses,retroviruses, lentiviruses, and rhabdoviruses.

The present invention is also applicable to any fungi, including yeasts,mycoplasma species, parasites, or other infectious organisms that causedisease in animals. Examples of fungi that can be treated with themethod and compositions of the present invention include fungi that growas molds or are yeastlike, including, for example, fungi that causediseases such as ringworm, histoplasmosis, blastomycosis, aspergillosis,cryptococcosis, sporotrichosis, coccidioidomycosis,paracoccidioidomycosis, mucormycosis, chromoblastomycosis,dermatophytosis, protothecosis, fusariosis, pityriasis, mycetoma,paracoccidioidomycosis, phaeohyphomycosis, pseudallescheriasis,sporotrichosis, trichosporosis, pneumocystis infection, and candidiasis.

The present invention can also be utilized to treat parasitic infectionsincluding, but not limited to, infections caused by tapeworms, such asTaenia, Hymenolepsis, Diphyllobothrium, and Echinococcus species,flukes, such as Fasciolopsis, Heterophyes, Metagonimus, Clonorchis,Fasciola, Paragonimus, and Schitosoma species, roundworms, such asEnterobius, Trichuris, Ascaris, Ancylostoma, Necator, Strongyloides,Trichinella, Wuchereria, Brugia, Loa Onchocerca, and Dracunculusspecies, ameba, such as Naegleria and Acanthamoeba species, andprotozoans, such as Plasmodium, Trypanosoma, Leishmania, Toxoplasma,Entamoeba, Giardia, Isospora, Cryptosporidium, and Enterocytozoonspecies.

The pathogenic cells to which the drug delivery conjugates of theinvention are directed can also be cells harboring endogenous pathogens,such as virus-, mycoplasma-, parasite-, or bacteria-infected cells, ifthese cells preferentially express vitamin receptors.

In one embodiment, the vitamin receptor binding drug delivery conjugatescan be internalized into the targeted pathogenic cells upon binding ofthe vitamin moiety to a vitamin receptor, transporter, or othersurface-presented protein that specifically binds the vitamin and whichis preferentially expressed on the pathogenic cells. Suchinternalization can occur, for example, through receptor-mediatedendocytosis. If the drug delivery conjugate contains a releasablelinker, the vitamin moiety and the drug can dissociate intracellularlyand the drug can act on its intracellular target.

In an alternate embodiment, the vitamin moiety of the drug deliveryconjugate can bind to the pathogenic cell placing the drug in closeassociation with the surface of the pathogenic cell. The drug can thenbe released by cleavage of the releasable linker. For example, the drugcan be released by a protein disulfide isomerase if the releasablelinker is a disulfide group. The drug can then be taken up by thepathogenic cell to which the vitamin receptor binding drug deliveryconjugate is bound, or the drug can be taken up by another pathogeniccell in close proximity thereto. Alternatively, the drug could bereleased by a protein disulfide isomerase inside the cell where thereleasable linker is a disulfide group. The drug may also be released bya hydrolytic mechanism, such as acid-catalyzed hydrolysis, as describedabove for certain beta elimination mechanisms, or by an anchimericallyassisted cleavage through an oxonium ion or lactonium ion producingmechanism. The selection of the releasable linker or linkers willdictate the mechanism by which the drug is released from the conjugate.It is appreciated that such a selection can be pre-defined by theconditions wherein the drug conjugate will be used.

In another embodiment, where the linker does not comprise a releasablelinker, the vitamin moiety of the drug delivery conjugate can bind tothe pathogenic cell placing the drug on the surface of the pathogeniccell to target the pathogenic cell for attack by other molecules capableof binding to the drug. Alternatively, in this embodiment, the drugdelivery conjugates can be internalized into the targeted cells uponbinding, and the vitamin moiety and the drug can remain associatedintracellularly with the drug exhibiting its effects withoutdissociation from the vitamin moiety.

In still another embodiment, or in combination with the above-describedembodiments, the vitamin receptor binding drug delivery conjugate canact through a mechanism independent of cellular vitamin receptors. Forexample, the drug delivery conjugates can bind to soluble vitaminreceptors present in the serum or to serum proteins, such as albumin,resulting in prolonged circulation of the conjugates relative to theunconjugated drug, and in increased activity of the conjugates towardsthe pathogenic cell population relative to the unconjugated drug.

In another embodiment of this invention, a vitamin receptor binding drugdelivery conjugate of the general formula V-L-D is provided. L isselected from (l_(s))_(a) and (l_(H))_(b), and combinations thereof,where (l_(s))_(a), (l_(H))_(b), and V are as defined herein, and D is adrug such as an immunogen. The immunogen can be a hapten, for example,fluorescein, dinitrophenyl, and the like. In this embodiment, thevitamin receptor binding drug delivery conjugate binds to the surface ofthe pathogenic cells and “labels” the cells with the immunogen, therebytriggering an immune response directed at the labeled pathogenic cellpopulation. Antibodies administered to the host in a passiveimmunization or antibodies existing in the host system from apreexisting innate or acquired immunity bind to the immunogen andtrigger endogenous immune responses. Antibody binding to the cell-boundvitamin-immunogen conjugate results in complement-mediated cytotoxicity,antibody-dependent cell-mediated cytotoxicity, antibody opsonization andphagocytosis, antibody-induced receptor clustering signaling cell deathor quiescence, or any other humoral or cellular immune responsestimulated by antibody binding to cell-bound ligand-immunogenconjugates. In cases where an immunogen can be directly recognized byimmune cells without prior antibody opsonization, direct killing of thepathogenic cells can occur. This embodiment is described in more detailin U.S. patent application Ser. No. 09/822,379, incorporated herein byreference. It is appreciated that in certain variations of thisembodiment where the drug is an immunogen, the bivalent linker may alsoinclude releasable linkers, as described above, such as a vitaminreceptor binding drug delivery conjugate of the general formula V-L-Dwhere L is selected from (l_(s))_(a); (l_(H))_(b), (l_(r))_(c), andcombinations thereof where (l_(s)) is a spacer linker, (l_(H)) is anheteroatom linker, (l_(r)) is a releasable linker, V is a vitamin, or ananalog or a derivative thereof, and a, b, and c are integers.

The vitamin receptor binding drug delivery conjugates described hereincomprise a vitamin receptor binding moiety, a bivalent linker (L), adrug, and, optionally, heteroatom linkers to link the vitamin receptorbinding moiety and the drug to the bivalent linker (L). The bivalentlinker (L) can comprise a spacer linker, a releasable (i.e., cleavable)linker, and an heteroatom linker, or combinations thereof.

The vitamin receptor binding drug delivery conjugates described hereincan be formed from a wide variety of vitamins or receptor-bindingvitamin analogs/derivatives, linkers, and drugs. The drug deliveryconjugates of the present invention are capable of selectively targetinga population of pathogenic cells in the host animal due to preferentialexpression of a receptor for the vitamin, accessible for vitaminbinding, on the pathogenic cells. Illustrative vitamin moieties includecarnitine, inositol, lipoic acid, pyridoxal, ascorbic acid, niacin,pantothenic acid, folic acid, riboflavin, thiamine, biotin, vitamin B₁₂,and the lipid soluble vitamins A, D, E and K. These vitamins, and theirreceptor-binding analogs and derivatives, constitute the targetingentity that can be coupled with the drug by bivalent linker (L) to formthe vitamin receptor binding drug delivery conjugates described herein.Therefore, the term “vitamin” includes vitamin analogs and/orderivatives (e.g., pteroic acid which is a derivative of folate, biotinanalogs such as biocytin, biotin sulfoxide, oxybiotin and other biotinreceptor-binding compounds, and the like). It should be appreciated thatin accordance with this invention, vitamin analogs or derivatives canmean a vitamin that incorporates an heteroatom through which the vitaminanalog or derivative is covalently bound to the bivalent linker (L).

Illustrative vitamin moieties include folic acid, biotin, riboflavin,thiamine, vitamin B₁₂, and receptor-binding analogs and derivatives ofthese vitamin molecules, and other related vitamin receptor bindingmolecules. Illustrative embodiments of vitamin analogs and/orderivatives include analogs and derivatives of folate such as folinicacid, pteropolyglutamic acid, and folate receptor-binding pteridinessuch as tetrahydropterins, dihydrofolates, tetrahydrofolates, and theirdeaza and dideaza analogs. The terms “deaza” and “dideaza” analogs referto the art-recognized analogs having a carbon atom substituted for oneor two nitrogen atoms in the naturally occurring folic acid structure,or analog or derivative thereof. For example, the deaza analogs includethe 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs of folate.The dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza,8,10-dideaza, and 5,8-dideaza analogs of folate. Other folates useful ascomplex forming ligands for this invention are the folatereceptor-binding analogs anminopterin, amethopterin (methotrexate),N¹⁰-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as1-deazamethopterin or 3-deazamethopterin, and3′,5′-dichloro-4-amino-4-deoxy-N¹⁰-methylpteroylglutamic acid(dichloromethotrexate). The foregoing folic acid analogs and/orderivatives are conventionally termed “folates,” reflecting theirability to bind with folate-receptors, and such ligands when conjugatedwith exogenous molecules are effective to enhance transmembranetransport, such as via folate-mediated endocytosis as described herein.Other suitable ligands capable of binding to folate receptors toinitiate receptor mediated endocytotic transport of the complex includeanti-idiotypic antibodies to the folate receptor. An exogenous moleculein complex with an anti-idiotypic antibody to a folate receptor is usedto trigger transmembrane transport of the complex in accordance with thepresent invention.

Illustrative embodiments of vitamin analogs and/or derivatives alsoinclude analogs and derivatives of biotin such as biocytin, biotinsulfoxide, oxybiotin and other biotin receptor-binding compounds, andthe like. It is appreciated that analogs and derivatives of the othervitamins described herein are also contemplated herein. In oneembodiment, vitamins that can be used in the drug delivery conjugatesdescribed herein include those that bind to vitamin receptors expressedspecifically on activated macrophages, such as the folate receptor,which binds folate, or an analog or derivative thereof as describedherein.

The binding site for the vitamin can include receptors for any vitaminmolecule, or a derivative or analog thereof, capable of specificallybinding to a receptor wherein the receptor or other protein is uniquelyexpressed, overexpressed, or preferentially expressed by a population ofpathogenic cells. A surface-presented protein uniquely expressed,overexpressed, or preferentially expressed by the pathogenic cells istypically a receptor that is either not present or present at lowerconcentrations on non-pathogenic cells providing a means for selectiveelimination of the pathogenic cells. The vitamin receptor binding drugdelivery conjugates may be capable of high affinity binding to receptorson cancer cells or other types of pathogenic cells. The high affinitybinding can be inherent to the vitamin moiety or the binding affinitycan be enhanced by the use of a chemically modified vitamin (i.e., ananalog or a derivative).

The drug can be any molecule capable of modulating or otherwisemodifying cell function, including pharmaceutically active compounds.Suitable molecules can include, but are not limited to: peptides,oligopeptides, retro-inverso oligopeptides, proteins, protein analogs inwhich at least one non-peptide linkage replaces a peptide linkage,apoproteins, glycoproteins, enzymes, coenzymes, enzyme inhibitors, aminoacids and their derivatives, receptors and other membrane proteins;antigens and antibodies thereto; haptens and antibodies thereto;hormones, lipids, phospholipids, liposomes; toxins; antibiotics;analgesics; bronchodilators; beta-blockers; antimicrobial agents;antihypertensive agents; cardiovascular agents includingantiarrhythmics, cardiac glycosides, antianginals and vasodilators;central nervous system agents including stimulants, psychotropics,antimanics, and depressants; antiviral agents; antihistamines; cancerdrugs including chemotherapeutic agents; tranquilizers;anti-depressants; H-2 antagonists; anticonvulsants; antinauseants;prostaglandins and prostaglandin analogs; muscle relaxants;anti-inflammatory substances; stimulants; decongestants; antiemetics;diuretics; antispasmodics; antiasthmatics; anti-Parkinson agents;expectorants; cough suppressants; mucolytics; and mineral andnutritional additives.

Further, the drug can be any drug known in the art which is cytotoxic,enhances tumor permeability, inhibits tumor cell proliferation, promotesapoptosis, decreases anti-apoptotic activity in target cells, is used totreat diseases caused by infectious agents, enhances an endogenousimmune response directed to the pathogenic cells, or is useful fortreating a disease state caused by any type of pathogenic cell. Drugssuitable for use in accordance with this invention includeadrenocorticoids and corticosteroids, alkylating agents, antiandrogens,antiestrogens, androgens, aclamycin and aclamycin derivatives,estrogens, antimetabolites such as cytosine arabinoside, purine analogs,pyrimidine analogs, and methotrexate, busulfan, carboplatin,chlorambucil, cisplatin and other platinum compounds, tamoxiphen, taxol,paclitaxel, paclitaxel derivatives, Taxotere®, cyclophosphamide,daunomycin, rhizoxin, T2 toxin, plant alkaloids, prednisone,hydroxyurea, teniposide, mitomycins, discodermolides, microtubuleinhibitors, epothilones, tubulysin, cyclopropyl benz[e]indolone,seco-cyclopropyl benz[e]indolone, O-Ac-seco-cyclopropyl benz[e]indolone,bleomycin and any other antibiotic, nitrogen mustards, nitrosureas,vincristine, vinblastine, and analogs and derivative thereof such asdeacetylvinblastine monohydrazide, colchicine, colchicine derivatives,allocolchicine, thiocolchicine, trityl cysteine, Halicondrin B,dolastatins such as dolastatin 10, amanitins such as α-amanitin,camptothecin, irinotecan, and other camptothecin derivatives thereof,geldanamycin and geldanamycin derivatives, estramustine, nocodazole,MAP4, colcemid, inflammatory and proinflammatory agents, peptide andpeptidomimetic signal transduction inhibitors, and any otherart-recognized drug or toxin. Other drugs that can be used in accordancewith the invention include penicillins, cephalosporins, vancomycin,erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycosideantibiotics, gentamicin, amphotericin B, acyclovir, trifluridine,ganciclovir, zidovudine, amantadine, ribavirin, and any otherart-recognized antimicrobial compound.

In one embodiment, the drugs for use in accordance with this inventionremain stable in serum for at least 4 hours. In another embodiment thedrugs have an IC₅₀ in the nanomolar range, and, in another embodiment,the drugs are water soluble. If the drug is not water soluble, thebivalent linker (L) can be derivatized to enhance water solubility. Theterm “drug” also means any of the drug analogs or derivatives describedhereinabove, including but not limited to the dolastatins such asdolastatin 10, the amanitins such as a-amanitin, camptothecins andirinotecans, and other camptothecin and irinotecan derivatives thereof.It should be appreciated that in accordance with this invention, a druganalog or derivative can mean a drug that incorporates an heteroatomthrough which the drug analog or derivative is covalently bound to thebivalent linker (L).

The vitamin receptor binding drug delivery conjugates of the presentinvention can comprise a vitamin receptor binding moiety, a bivalentlinker (L), a drug, and, optionally, heteroatom linkers to link thevitamin receptor binding moiety and the drug to the bivalent linker (L).It should be appreciated that in accordance with this invention, avitamin analog or derivative can mean a vitamin that incorporates anheteroatom through which the vitamin analog or derivative is covalentlybound to the bivalent linker (L). Thus, the vitamin can be covalentlybound to the bivalent linker (L) through an heteroatom linker, or avitamin analog or derivative (i.e., incorporating an heteroatom) can bedirectly bound to the bivalent linker (L). Similarly, a drug analog orderivative is a drug in accordance with the invention and a drug analogor derivative can mean a drug that incorporates an heteroatom throughwhich the drug analog or derivative is covalently bound to the bivalentlinker (L). Thus, the drug can be covalently bound to the bivalentlinker (L) through an heteroatom linker, or a drug analog or derivative(i.e., incorporating an heteroatom) can be directly bound to thebivalent linker (L). The bivalent linker (L) can comprise a spacerlinker, a releasable (i.e., cleavable) linker, and an heteroatom linkerto link the spacer linker to the releasable linker in conjugatescontaining both of these types of linkers.

Thus, in accordance with this invention the bivalent linker (L) cancomprise a means for associating the vitamin with the drug, such as byconnection through heteroatom linkers (i.e., spacer arms or bridgingmolecules), or by direct covalent bonding of the bivalent linker (L) toa vitamin or drug analog or derivative. Either means for associationshould not prevent the binding of the vitamin, or vitamin receptorbinding derivative or analog, to the vitamin receptor on the cellmembrane for operation of the method of the present invention.

Generally, any manner of forming a conjugate between the bivalent linker(L) and the vitamin, or analog or derivative thereof, between thebivalent linker (L) and the drug, or analog or derivative thereof,including any intervening heteroatom linkers, can be utilized inaccordance with the present invention. Also, any art-recognized methodof forming a conjugate between the spacer linker, the releasable linker,and the heteroatom linker to form the bivalent linker (L) can be used.The conjugate can be formed by direct conjugation of any of thesemolecules, for example, through hydrogen, ionic, or covalent bonds.Covalent bonding can occur, for example, through the formation of amide,ester, disulfide, or imino bonds between acid, aldehyde, hydroxy, amino,sulfhydryl, or hydrazo groups.

The spacer and/or releasable linker (i.e., cleavable linker) can be anybiocompatible linker. The cleavable linker can be, for example, a linkersusceptible to cleavage under the reducing or oxidizing conditionspresent in or on cells, a pH-sensitive linker that may be an acid-labileor base-labile linker, or a linker that is cleavable by biochemical ormetabolic processes, such as an enzyme-labile linker. Typically, thespacer and/or releasable linker comprises about 1 to about 30 carbonatoms, more typically about 2 to about 20 carbon atoms. Lower molecularweight linkers (i.e., those having an approximate molecular weight ofabout 30 to about 300) are typically employed. Precursors to suchlinkers are typically selected to have either nucleophilic orelectrophilic functional groups, or both, optionally in a protected formwith a readily cleavable protecting group to facilitate their use insynthesis of the intermediate species.

The invention is also directed to pharmaceutical compositions comprisingan amount of a vitamin receptor binding drug delivery conjugateeffective to eliminate a population of pathogenic cells in a host animalwhen administered in one or more doses. The drug delivery conjugate ispreferably administered to the host animal parenterally, e.g.,intradermally, subcutaneously, intramuscularly, intraperitoneally,intravenously, or intrathecally. Alternatively, the drug deliveryconjugate can be administered to the host animal by other medicallyuseful processes, such as orally, and any effective dose and suitabletherapeutic dosage form, including prolonged release dosage forms, canbe used.

Examples of parenteral dosage forms include aqueous solutions of theactive agent, in an isotonic saline, 5% glucose or other well-knownpharmaceutically acceptable liquid carriers such as liquid alcohols,glycols, esters, and amides. The parenteral dosage form in accordancewith this invention can be in the form of a reconstitutable lyophilizatecomprising the dose of the drug delivery conjugate. In one aspect of thepresent embodiment, any of a number of prolonged release dosage formsknown in the art can be administered such as, for example, thebiodegradable carbohydrate matrices described in U.S. Pat. Nos.4,713,249; 5,266,333; and 5,417,982, the disclosures of which areincorporated herein by reference, or, alternatively, a slow pump (e.g.,an osmotic pump) can be used.

At least one additional composition comprising a therapeutic factor canbe administered to the host in combination or as an adjuvant to theabove-detailed methodology, to enhance the drug deliveryconjugate-mediated elimination of the population of pathogenic cells, ormore than one additional therapeutic factor can be administered. Thetherapeutic factor can be selected from a compound capable ofstimulating an endogenous immune response, a chemotherapeutic agent, oranother therapeutic factor capable of complementing the efficacy of theadministered drug delivery conjugate. The method of the invention can beperformed by administering to the host, in addition to theabove-described conjugates, compounds or compositions capable ofstimulating an endogenous immune response (e.g. a cytokine) including,but not limited to, cytokines or immune cell growth factors such asinterleukins 1-18, stem cell factor, basic FGF, EGF, G-CSF, GM-CSF,FLK-2 ligand, HILDA, MIP-1α, TGF-α, TGF-β, M-CSF, IFN-α, IFN-β, IFN-γ,soluble CD23, LIF, and combinations thereof.

Therapeutically effective combinations of these factors can be used. Inone embodiment, for example, therapeutically effective amounts of IL-2,for example, in amounts ranging from about 0.1 MIU/m²/dose/day to about15 MIU/m²/dose/day in a multiple dose daily regimen, and IFN-α, forexample, in amounts ranging from about 0.1 MIU/m²/dose/day to about 7.5MIU/m²/dose/day in a multiple dose daily regimen, can be used along withthe drug delivery conjugates to eliminate, reduce, or neutralizepathogenic cells in a host animal harboring the pathogenic cells(MIU=million international units; m²=approximate body surface area of anaverage human). In another embodiment IL-12 and IFN-α are used in theabove-described therapeutically effective amounts for interleukins andinterferons, and in yet another embodiment IL-15 and IFN-α are used inthe above described therapeutically effective amounts for interleukinsand interferons. In an alternate embodiment IL-2, IFN-α or IFN-γ, andGM-CSF are used in combination in the above described therapeuticallyeffective amounts. The invention also contemplates the use of any othereffective combination of cytokines including combinations of otherinterleukins and interferons and colony stimulating factors.

Chemotherapeutic agents, which are, for example, cytotoxic themselves orcan work to enhance tumor permeability, are also suitable for use in themethod of the invention in combination with the drug deliveryconjugates. Such chemotherapeutic agents include adrenocorticoids andcorticosteroids, alkylating agents, antiandrogens, antiestrogens,androgens, aclamycin and aclamycin derivatives, estrogens,antimetabolites such as cytosine arabinoside, purine analogs, pyrimidineanalogs, and methotrexate, busulfan, carboplatin, chlorambucil,cisplatin and other platinum compounds, tamoxiphen, taxol, paclitaxel,paclitaxel derivatives, Taxotere®, cyclophosphamide, daunomycin,rhizoxin, T2 toxin, plant alkaloids, prednisone, hydroxyurea,teniposide, mitomycins, discodermolides, microtubule inhibitors,epothilones, tubulysin, cyclopropyl benz[e]indolone, seco-cyclopropylbenz[e]indolone, O-Ac-seco-cyclopropyl benz[e]indolone, bleomycin andany other antibiotic, nitrogen mustards, nitrosureas, vincristine,vinblastine, and analogs and derivative thereof such asdeacetylvinblastine monohydrazide, colchicine, colchicine derivatives,allocolchicine, thiocolchicine, trityl cysteine, Halicondrin B,dolastatins such as dolastatin 10, amanitins such as α-amanitin,camptothecin, irinotecan, and other camptothecin derivatives thereof,geldanamycin and geldanamycin derivatives, estramustine, nocodazole,MAP4, colcemid, inflammatory and proinflammatory agents, peptide andpeptidomimetic signal transduction inhibitors, and any otherart-recognized drug or toxin. Other drugs that can be used in accordancewith the invention include penicillins, cephalosporins, vancomycin,erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycosideantibiotics, gentamicin, amphotericin B, acyclovir, trifluridine,ganciclovir, zidovudine, amantadine, ribavirin, maytansines and analogsand derivatives thereof, gemcitabine, and any other art-recognizedantimicrobial compound.

The therapeutic factor can be administered to the host animal prior to,after, or at the same time as the vitamin receptor binding drug deliveryconjugates and the therapeutic factor can be administered as part of thesame composition containing the drug delivery conjugate or as part of adifferent composition than the drug delivery conjugate. Any suchtherapeutic composition containing the therapeutic factor at atherapeutically effective dose can be used in the present invention.

Additionally, more than one type of drug delivery conjugate can be used.For example, the host animal can be treated with conjugates withdifferent vitamins, but the same drug (e.g., folate-mitomycin conjugatesand vitamin B₁₂-mitomycin conjugates) in a co-dosing protocol. In otherembodiments, the host animal can be treated with conjugates comprisingthe same vitamin linked to different drugs, or various vitamins linkedto various drugs. For example, the host animal could be treated with afolate-mitomycin and a folate-cisplatin conjugate, or with afolate-mitomycin conjugate and a vitamin B₁₂-cisplatin conjugate.Furthermore, drug delivery conjugates with the same or differentvitamins, and the same or different drugs comprising multiple vitaminsand multiple drugs as part of the same drug delivery conjugate could beused.

The unitary daily dosage of the drug delivery conjugate can varysignificantly depending on the host condition, the disease state beingtreated, the molecular weight of the conjugate, its route ofadministration and tissue distribution, and the possibility of co-usageof other therapeutic treatments such as radiation therapy. The effectiveamount to be administered to a patient is based on body surface area,patient weight, and physician assessment of patient condition. Effectivedoses can range, for example, from about 1 ng/kg to about 1 mg/kg, fromabout 1 μg/kg to about 500 μg/kg, and from about 1 μg/kg to about 100μg/kg.

Any effective regimen for administering the drug delivery conjugates canbe used. For example, the drug delivery conjugates can be administeredas single doses, or can be divided and administered as a multiple-dosedaily regimen. Further, a staggered regimen, for example, one to threedays per week can be used as an alternative to daily treatment, and forthe purpose of defining this invention such intermittent or staggereddaily regimen is considered to be equivalent to every day treatment andwithin the scope of this invention. In one embodiment of the inventionthe host is treated with multiple injections of the drug deliveryconjugate to eliminate the population of pathogenic cells. In oneembodiment, the host is injected multiple times (preferably about 2 upto about 50 times) with the drug delivery conjugate, for example, at12-72 hour intervals or at 48-72 hour intervals. Additional injectionsof the drug delivery conjugate can be administered to the patient at aninterval of days or months after the initial injections(s) and theadditional injections prevent recurrence of the disease state caused bythe pathogenic cells.

In one embodiment, vitamins, or analogs or derivatives thereof, that canbe used in the drug delivery conjugates of the present invention includethose that bind to receptors expressed specifically on activatedmacrophages, such as the folate receptor which binds folate, or ananalog or derivative thereof. The folate-linked conjugates, for example,can be used to kill or suppress the activity of activated macrophagesthat cause disease states in the host. Such macrophage targetingconjugates, when administered to a patient suffering from an activatedmacrophage-mediated disease state, work to concentrate and associate theconjugated drug in the population of activated macrophages to kill theactivated macrophages or suppress macrophage function. Elimination,reduction, or deactivation of the activated macrophage population worksto stop or reduce the activated macrophage-mediated pathogenesischaracteristic of the disease state being treated. Exemplary of diseasesknown to be mediated by activated macrophages include rheumatoidarthritis, ulcerative colitis, Crohn's disease, psoriasis,osteomyelitis, multiple sclerosis, atherosclerosis, pulmonary fibrosis,sarcoidosis, systemic sclerosis, organ transplant rejection (GVHD) andchronic inflammations. Administration of the drug delivery conjugate istypically continued until symptoms of the disease state are reduced oreliminated.

The drug delivery conjugates administered to kill activated macrophagesor suppress the function of activated macrophages can be administeredparenterally to the animal or patient suffering from the disease state,for example, intradermally, subcutaneously, intramuscularly,intraperitoneally, or intravenously in combination with apharmaceutically acceptable carrier. Alternatively, the drug deliveryconjugates can be administered to the animal or patient by othermedically useful procedures and effective doses can be administered instandard or prolonged release dosage forms. The therapeutic method canbe used alone or in combination with other therapeutic methodsrecognized for treatment of disease states mediated by activatedmacrophages.

The following drug delivery conjugates are illustrative of drug deliveryconjugates that are contemplated to fall within the scope of theinvention described herein. These drug delivery conjugates can beprepared in accordance with the invention using the procedures describedherein in addition to art-recognized protocols.

In addition, the following drug delivery conjugates are alsoillustrative of drug delivery conjugates that are contemplated to fallwithin the scope of the invention described herein. Theaccompanying-synthetic procedures are illustrative of those that can beused to prepare the drug delivery conjugates described herein.

To a solution of diacetoxyscirpenol (DAS) in acetonitrile is added 1.0eg. of 1,2,4,5-benzenetetracarboxylic dianhydride followed by 1 eg. ofHünig's base. The reaction mixture is stirred for 1.5 hours under argonand at room temperature. If some DAS remains unreacted, an additional0.2 eg. of dianhydride is added and stirring is continued for 1 hour. Asolution of 1.2 eg. of pteroyl hydrazide (prepared according to J. Am.Chem. Soc., 1997, 19, 10004, the disclosure of which is incorporatedherein by reference) in anhydrous DMSO is added, followed by 1.0 eg. ofHünig's base. The reaction mixture is stirred for 1 hour andprecipitated in diethyl ether. The resulting precipitate is furtherpurified by preparative HPLC.

As generally described in Example 10a. Boc-hydrazide is reacted withsuccinic anhydride, and the resulting product is reacted with5″-amino-bis-indolyl-seco-CBI in the presence of EDC as condensingagent. Boc removal and acyl hydrazone formation with free levulinic acidfurnishs, after NHS-ester activation, a reactive partner for the peptidefragment Pte-γ-Glu-Asp-Arg-Asp-Dap-OH. This peptide fragment is preparedby a polymer-supported sequential approach using the Fmoc-strategystarting with Fmoc-Dap(Boc)-Wang resin,as generally described in Scheme12.

Fmoc-hydrazide is reacted with 3-(2-pyridyldithio)propionic acid to giveFmoc-hydrazido-[3-(2-pyridyldithio)propionate]. Reaction with amitomycin C derivative (mitomycin C, N-(CH₂)₂SH) results in adisulfide-containing derivative of mitomycin C. Fmoc-removal usingstandard protocols and acyl hydrazone formation with levulinic acidprovides, after consecutive NHS-ester activation, a reactive partner forthe peptide fragment Pte-γ-Glu-Dap-OH, which may be prepared asgenerally described in Scheme 12.

The following illustrative exemplified embodiments are are not intendedand should not be construed as limiting. For example, in each compoundpresented herein, the stereochemistry of amino acids used in forming thelinker may be optionally selected from the natural L configuration, orthe unnatural D configuration. Each Example was characterized by NMR,MS, and/or UV spectroscopy, and/or HPLC as indicated; selectedcharacteristic signals are noted as appropriate.

EXAMPLE 1

Diethylenetriaminefolic acid, γ-amide (DETA-folate) was synthesizedaccording to a procedure described by P. Fuchs et al., J. Am. Chem.Soc., 1997, 119, 10004, the disclosure of which is incorporated hereinby reference. This compound (100 mg) was dissolved in 2 mL of 0.1 N HCl.The resulting solution was added to a solution of K₂PtCl₄ (158 mg) in 1mL of 0.1 N HCl with stirring. Three mL of DMSO were added and stirringwas continued for 3 days, the solution was filtered, and the filtrateprecipitated in acetonitrile to give 170 mg of a yellow powder; MS(MALDI) 1249.92, 1286.27; ¹H NMR (D₂O) δ 1.05 (t, 1H), 2.3 (t, 2H), 3.1(t, 2H), 4.45 (m, 1H), 6.65 (d, 2H), 7.5 (d, 2H), 8.65 (s, 1H).

EXAMPLE 2a

The N¹⁰-trifluoroacetyl protected folate-containing peptidyl fragmentN¹⁰-TFA-Pte-Glu-Glu-Lys-OH was prepared by a polymer-supportedsequential approach using the Fmoc-strategy, as generally illustrated inScheme 12. It was synthesized on the acid-sensitive Fmoc-Lys(Boc)-Wangresin. PyBop was applied as the activating reagent to ensure efficientcoupling using low equivalents of amino acids. Fmoc protecting groupswere removed after every coupling step under standard conditions (20%piperidine in DMF). Fmoc-Glu-OtBu and N¹⁰-TFA-Pte-OH were used asprotected amino acid building blocks. After the last assembly step thepeptide was cleaved from the polymeric support by treatment withtrifluoroacetic acid, ethanedithiol and triisopropylsilane. Thisreaction also resulted in simultaneous removal of the t-Bu and t-Bocprotecting groups. The crude peptide was purified by preparative HPLC togive N¹⁰-TFA-Pte-γGlu-γGlu-Lys-OH as a TFA salt. A solution of 81 mg(0.1 mmol) of the peptide in 2 mL DMSO was treated with 15 μL (0.11mmol) Et₃N and 35 mg (0.1 mmol) of mitomycin A. Mitomycin A may beprepared from Mitomycin C according to the procedures of M. Matsui, Y.Yamada, K. Uzu, and T. Hirata, J. Antibiot. 21, 189-198 (1968) and D.Vias, D. Benigni, R. Partyka, and T. Doyle, J. Org. Chem. 51, 4307-4309(1986), the disclosures of which are incorporated herein by reference.The reaction mixture was stirred for 48 h at room temperature and thesolvent removed by freeze-drying. Unless otherwise noted, allevaporations of solvent were conducted under reduced pressure. Finally,the trifluoroacetyl protective group was detached in aqueous ammoniumhydroxide (pH=10.0) and the product was precipitated in acetonitrile togive 102 mg of the conjugate as a yellow solid; ¹H NMR (D₂O) δ 2.45 (q,1H), 2.95 (m, 2H), 3.35 (dd, 1H), 3.5 (d, 1H), 6.5 (d, 2H), 7.55 (d,2H), 8.55 (s, 1H).

EXAMPLE 2b

The N¹⁰-trifluoroacetyl protected folate-containing peptidyl fragmentN¹⁰-TFA-Pte-Glu-Cys-OH was prepared by a polymer-supported sequentialapproach using the Fmoc-strategy, as generally illustrated in Scheme 12,and described in Example 2a. Cystamine was reacted with mitomycin A (seeMatsui et al., J. Antibiot. 21, 189-198 (1968); Vias et al., J. OrgChem. 51, 4307-4309 (1986)) giving the disulfide containing mitomycin Cderivative possessing a terminal free amino group, which was coupledwith levulinic acid, followed by subsequent reaction of the carbonylgroup with a maleinido derivatized acyl hydrazide. Reaction of theresulting Michael acceptor with N¹⁰-TFA-Pte-Glu-Cys-OH gave the finalconjugate after removal of the trifluoroacetyl protective group withaqueous ammonium hydroxide (pH=10.0), and precipitation fromacetonitrile; MS (MALDI) 1059.04, 1148.44, 1225.32, 1300.8; ¹H NMR (D₂O)δ 1.8 (d, 2H), 1.9 (s, 1H), 2.3 (q, 1h), 2.45 (q, 1H), 2.9 (t, 1H), 3.35(dd, 1H), 4.45 (s, 1H), 4.5 (dd, 1H), 6.65 (d, 2H), 7.55 (d, 2H), 8.6(s, 1H).

EXAMPLE 3

The intermediate maleimido p-methoxybenzylidene acetal of T-2 toxin wassynthesized starting from commercially availableN-(2-hydroxyethyl)maleimide. Its hydroxyl group was reacted withp-methoxybenzyl chloride (1.2 eg.) in the presence of silver(I) oxide (2eg.) as a mild base in methylene chloride. The crude product waspurified on a Silica column. Oxidative treatment of the resultingp-methoxybenzyl ether with 1.5 eg. of2,3-dichloro-5,6-dicyano-benzoquinone (DDQ) in the presence of theOH-containing T-2 toxin (1 eg.) gave the desired p-methoxybenzylideneacetal via the stabilized p-methoxybenzylcarbenium intermediate species.

The other reaction partner, Pte-γ-Glu-Arg-Asp-Cys-OH, was prepared by apolymer-supported sequential approach using the Fmoc-strategy. It wassynthesized on the acid-sensitiveH-Cys(4-methoxytrityl)-2-chlorotrityl-resin. PyBop was applied as theactivating reagent to ensure efficient coupling using low equivalents ofamino acids. Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu-OtBu), andN¹⁰-TFA-Pte-OH were used as protected amino acid building blocks. Fmocprotecting groups were removed after every coupling step under standardconditions (20% piperidine in DMF). After the last assembly step thepeptide was cleaved from the polymeric support by treatment withtrifluoroacetic acid, ethanedithiol and triisopropylsilane. Thisreaction also resulted in simultaneous removal of the t-Bu and t-Bocprotecting groups. The crude peptide was purified by preparative HPLC togive N¹⁰-TFA-Pte-γ-Glu-Arg-Asp-Cys-OH. The trifluoroacetyl protectivegroup was detached in aqueous ammonium hydroxide (pH=10.0).

Finally, the targeted p-methoxybenzylidene acetal-tethered folate-drugconjugate was prepared by mixing under argon a buffered water solution(pH=7.0) of the peptide with an equimolar acetonitrile solution of themaleimido-containing acetal of T-2 toxin. After stirring at roomtemperature for 1 hour the final conjugate was subjected to preparativeHPLC and gave a yellow powder after freeze-drying of the collectedfraction; MS (m+H)⁺ 1541.3; ¹H NMR (DMSO-d₆) δ 0.1 (s, 1H), 0.55 (d,2H), 0.9 (dd, 3H), 1.65 (s, 1H), 2.0 (d, 1H), 3.75 (d, 2H), 5.25 (d,1H), 6.65 (d, 2H), 6.9 (d, 2H), 7.3 (t, 2H), 7.65 (d, 2H), 8.65 (s, 1H).

EXAMPLE 4a

EXAMPLE 4b

EXAMPLE 4c

The compounds of Examples 4a, 4b, and 4c were prepared according to theprocedure generally described in Example 3, except that theacylaziridine was prepared by acylation (see Scheme 1) of mitomycin Awith the appropriate commercially available N-(alkanoic acid)maleimide.

EXAMPLE 5

Reaction of trans-4-aminocyclohexanol hydrochloride with an equimolaramount of Fmoc-OSu in the presence of 2.2 eg. of NaHCO₃ as a base, andacetonitrile/water (1/1) as a solvent gave N-Fmoc-protected aminoalcoholwhich was oxidized to the corresponding N-Fmoc-protected aminoketoneapplying Swern's conditions (Synthesis, 1981, 165). Ketalization with 4eq. of methyl orthoformate and a catalytic amount of trifluoroaceticacid gave an N-Fmoc-protected aminoketal in quantitative yield.Treatment of this ketal with equimolar amounts of trimethylsilyltrifluoromethanesulfonate and 2,4,6-tri-t-butyl-pyridine resulted in4-Fmoc-aminocyclohexyl enol ether as a product. In the next step, thedrug, T-2 toxin, was treated with a four-fold excess of the enol etherin the presence of molecular sieves (3 Å) and catalytic amounts oftrifluoroacetic acid. The resulting unsymmetrical mixed ketal waspurified on silica. The Fmoc protective group was removed by treatmentwith resin-bound piperidine in DMF. The liberated amino group wasreacted with 1.1 eg. of maleimidoacetic acid-NHS-ester in the presenceof 1.1 eg. of Hünig's base. The maleimido-containing ketal of T-2 toxinwas purified on silica.

The folate-containing peptide fragment, Pte-γ-Glu-β-Dap-Asp-Cys-OH, wasprepared by a polymer-supported sequential approach using theFmoc-strategy, as generally described in Scheme 12. It was synthesizedon the acid-sensitive Wang resin loaded with Fmoc-L-Cys(Trt)-OH. PyBopwas applied as the activating reagent to ensure efficient coupling usinglow equivalents of amino acids. Fmoc protecting groups were removedafter every coupling step under standard conditions (20% piperidine inDMF). Fmoc-Asp(OtBu)-OH, Boc-Dap(Fmoc)-OH, Fmoc-Glu-OtBu, andN¹⁰-TFA-Pte-OH were used as protected amino acid building blocks. Afterthe last assembly step the peptide was cleaved from the polymericsupport by treatment with trifluoroacetic acid, ethanedithiol andtriisopropylsilane. This reaction also resulted in simultaneous removalof the t-Bu, t-Boc, and trityl protecting groups. Finally, thetrifluoroacetyl moiety was detached in aqueous ammonium hydroxide togive the desired thiol-containing peptide. The crude peptide waspurified by preparative HPLC.

Finally, the targeted ketal-tethered folate-drug conjugate was preparedby mixing under argon buffered water solution (pH=7.0) the peptide withan equimolar acetonitrile solution of the maleimido-containing ketal ofT-2 toxin. After stirring at room temperature for 1 hour the finalconjugate was subjected to preparative HPLC and gave a yellow powderafter freeze-drying of the collected fraction; ES MS (m−H)⁻ 1474.5,(m+H)⁺ 1476.2, (m+Na)⁺ 1498.3.

EXAMPLE 6

Ethylenediaminefolic acid, γ-amide (EDA-folate) was synthesizedaccording to a procedure described by P. Fuchs et al. (J. Am. Chem.Soc., 1997, 119, 10004; see e.g. synthesis of compound 52 describedtherein), the disclosure of which is incorporated herein by reference.EDA-folate (600 mg) was suspended in 5 mL of anhydrous DMSO. Afterstirring for 4 hours at 60° C. the resulting solution was cooled to 20°C. and 4 eg. of 1,2,4,5-benzenetetracarboxylic dianhydride (BTCAanhydride) was added. After 5 min the reaction-mixture was poured intowell-stirred anhydrous acetonitrile. The resulting precipitate wasisolated by centrifugation to give 657 mg ofBTCA(monoanhydride)-EDA-folate.

To a well-stirred solution of daunomycin in dry DMSO was added solidBTCA(monoanhydride)-EDA-folate (1.5 eg.). After stirring for anadditional 14 hours, 50% of the daunomycin remained unreacted (HPLC),and 1.5 eg. of BTCA(monoanhydride)-EDA-folate were then added. Afterstirring for and additional four hours all of the daunomycin wasconsumed. In the HPLC profile two new peaks were observed with closeretention times representing the two regioisomers of the finalconjugate. The crude product was isolated after precipitation inacetonitrile and was further purified on reverse phase HPLC. Thestructure of the product was in agreement with the ES MS (m−H)⁻ 1227.1.

EXAMPLE 7

Under argon and at 0° C. to a well-stirred solution of 250 mg (0.25mmol) of paclitaxel and 130 μL (0.73 mmol) of Hünig's base in 4 mL ofanhydrous dichloromethane were added slowly 85 μL (0.8 mmol) Alloc-Cl.The stirring was continued for an additional 12 hours and the productwas isolated using standard extraction techniques. This white powder,2′-alloc-paclitaxel, was used in the next step without furtherpurification.

In this step, 108 mg (0.117 mmol) of 2′-alloc-paclitaxel were dissolvedin 1.0 mL of anhydrous acetonitrile. Under argon with stirring, 25 mg(0.117 mmol) of 1,2,4,5-benzenetetracarboxylic dianhydride (BTCAanhydride) and 21 μL (0.120 mmol) of Hünig's base were added to thissolution. The stirring was continued for an additional 2.5 hours. In aseparate reaction flask 52 mg of EDA-folate were stirred at 60° C. untilall material was dissolved (ca. 60 min). After cooling to roomtemperature, the previous reaction mixture was added to this solutionand stirring was continued for an additional 2 hours. The reactionmixture was added drop-wise to a well-stirred mixture ofacetonitrile/diethyl ether (20:80). The yellow precititate was separatedby centrifugation and further purified by preparative HPLC. Thestructure of the product was in agreement with the 1D and 2D (COSY)¹H-NMR spectra; ES MS (m+H)⁺ 1555.5.

EXAMPLE 8

A mixture of 1.0 eg. of aclamycin, 2.0 eg. ofhydrazido-[3-(2-pyridyldithio)propionate] (SPDP-hydrazone) and a fewcrystals of pyridinium p-toluenesulfonate were dissolved under argonwith stirring in anhydrous methanol. The reaction mixture was stirred atroom temperature for 8 hours. The solvent was evaporated to dryness. Theresidue was purified on a silica column pretreated with 1.5%triethylamine in chloroform/methanol (90:10). The aclamycin acylhydrazone obtained was dissolved in a minimal amount of acetonitrile. Tothe resulting solution was added slowly and under argon an equimolaramount of Pte-γ-Glu-Cys-OH (dissolved in water and adjusted to pH=6.8).The preparation of Pte-γ-Glu-Cys-OH is analogous to the proceduredescribed in Example 2a, and generally described in Scheme 12. Thedisulfide exchange reaction took place in 10 min. The reaction mixturewas added slowly to an excess of acetonitrile and the resultingprecipitate was isolated after centrifugation. The precipitate wasresuspended once more in acetonitrile and after stirring for 15 min wasseparated by centrifugation. After drying under high vacuum over nightthe final conjugate was sufficiently pure (HPLC); ES MS (m+H)⁺ 1474.1.

EXAMPLE 9

A mixture of 1.0 eg. of aclamycin and 1.2 eg. of β-maleimidopropionicacid.TFA was dissolved under argon with stirring in anhydrous methanol.The reaction mixture was stirred at room temperature for 1 hour. Thesolvent was evaporated to dryness. The residue was passed through ashort silica column pretreated with 1.5% triethylamine inchloroform/methanol (90:10). In a separate flask, the peptide fragmentPte-γ-Glu-γ-Glu Cys-OH was dissolved in water under argon whileadjusting the pH to 6.8. The preparation of Pte-γ-Glu-γ-Glu-Cys-OH isanalogous to the procedure described in Example 2a, and generallydescribed in Scheme 12. To the resulting yellowish solution was addedslowly the maleimido-hydrazone of aclamycin dissolved in a minimalamount of methanol. The reaction mixture was stirred for 1 hour underargon. The methanol was removed and the residue purified by HPLC on apreparative column, followed by freeze-drying; ES MS (m+H)⁺ 1722.3.

EXAMPLE 9b

EXAMPLE 9c

The compounds of Examples 9b and 9c were prepared from doxorubicin(14-hydroxydaunomycin) derivatives according to the procedure generallydescribed in Example 9a.

EXAMPLE 10a

The 5″-(N-Boc)amino analog of the highly potent cytotoxic drugbis-indolyl-seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one(bis-indolyl-seco-CBI) was prepared according to a slightly modifiedprocedure described first by D. Boger et al., J. Org. Chem., 1992, 57,2873, the disclosure of which is incorporated herein by reference.

The peptide fragment, Pte-γ-Glu-Asp-Arg-Asp-Cys-OH, was prepared by apolymer-supported sequential approach using the Fmoc-strategy on theacid- sensitive H-Cys(4-methoxytrityl)-2-chlorotrityl-resin, asgenerally described in Scheme 12. PyBop was applied as the activatingreagent to ensure efficient coupling using low equivalents of aminoacids. Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu-OtBu), andN¹⁰-TFA-Pte-OH were used as protected amino acid building blocks. Fmocprotecting groups were removed after every coupling step under standardconditions (20% piperidine in DMF). After the last assembly step thepeptide was cleaved from the polymeric support by treatment withtrifluoroacetic acid, ethanedithiol and triisopropylsilane. Thisreaction also resulted in simultaneous removal of the t-Bu and t-Bocprotecting groups. The crude peptide was purified by preparative HPLC togive N¹⁰-TFA-Pte-γ-Glu-Asp-Arg-Asp-Cys-OH. The trifluoroacetylprotective group was detached in aqueous ammonium hydroxide (pH=10.0).

Lev-Val-OH was synthesized using a standard protocol includingcondensation of valine methyl ester hydrochloride with levolinic acid inthe presence of EDC and Hünig's base followed by hydrolysis of themethyl ester with lithium hydroxide and water.

The final assembly of the complex conjugate started with the removal ofthe N-Boc group from 5″-(N-Boc)amino-bis-indolyl-seco-CBI and couplingof the liberated amino group with the carboxy functionality ofLev-Val-OH in the presence of EDC. Acyl hydrazone formation wasaccomplished by the reaction of the ketone functionality of thelevolinic moiety with 1.2 eg. of β-maleimidopropionic acid.TFA intetrahydrofuran. After chromatographic purification (silica,TBF/hexane=1/1) the product of the above reaction was dissolved in DMSO.To this solution under argon was added 0.9 eg. ofPte-γ-Glu-Asp-Arg-Asp-Cys-OH and the reaction mixture was stirred for 18hours. The solvent was removed by freeze-drying and the residue waspurified by HPLC.

EXAMPLE 10b

EXAMPLE 10c

The compounds of Examples 10c and 10c were prepared from derivatives of5″-(N-Boc)amino-bis-indolyl-seco-CBI according to the proceduregenerally described in Example 10a.

EXAMPLE 11

In the presence of potassium carbonate, S-alkylation of2-mercaptoethanol was accomplished with allyl bromide. The hydroxylgroup in the resulting allyl β-hydroxyethyl sulfide was exchanged forchlorine with thionyl chloride. Oxidation of this product with hydrogenperoxide in the presence of acetic acid and acetic anhydride (J. Am.Chem. Soc., 1950, 72, 59, the disclosure of which is incorporated hereinby reference) resulted in allyl β-chloroethyl sulfone. Reaction of thisproduct with chlorodimethylsilane in the presence of a catalytic amountof hydrogen hexachloroplatinate(IV) and with elevated temperature gave3-(β-chloroethyl sulfonyl)propyl dimethylsilyl chloride afterdistillation. This chlorosilane silylated the hydroxyl group of thehighly cytotoxic compound rhizoxin while using 1 eg. of pyridine as abase. Treatment of the β-chloroethyl sulfone moiety in this moleculewith excess of triethylamine resulted in a smooth β-elimination ofhydrogen chloride with formation of the respective vinyl sulfone.

The other reaction partner, the peptide fragmentPte-γ-Glu-Arg-Asp-Cys-OH, was prepared by a polymer-supported sequentialapproach using an Fmoc protocol, as generally described in Example 2aand Scheme 12.

The final assembly of the complex conjugate was achieved by Michaeladdition of the thiol group of the peptide fragment to the vinyl sulfonemoiety of the silicon linker attached to rhizoxin. Reaction medium forthis transformation was 50:50 acetonitrile/water (pH=7.2). Afterstirring at room temperature for 24 hours, the final conjugate wasisolated after HPLC on a preparative column; ES MS (m+H)⁺ 1631.6; (m−H)⁻1629.6.

EXAMPLE 12

This silicon-tethered conjugate of rhizoxin was synthesized using theprotocol described in Example 11, except that commercially availablechloromethylphenylsilane was used instead of chlorodimethylsilane.

EXAMPLE 13 General Preparation of Compounds Containing a CysteineDisulfide Bond

Thiosulfonates 4 (1 eq.), prepared according to the method of Ranasingheand Fuchs, Synth. Commun. 18(3), 227-32 (1988), the disclosure of whichis incorporated herein by reference, are reacted with drugs, druganalogs, or drug derivatives 5 (1 eq.) to prepare the drugthiosulfonates 6 as a solution in methanol, as shown in Scheme 13. R isalkyl or aryl, L is a suitable leaving group, such as halogen,pentafluorophenyl, and the like, n is an integer from 1 to 4, and X is—O—, —NH—, —C(O)O—, or —C(O)NH—. Conversion is conveniently monitored byobserving the disappearance of each starting material by TLC (silicagel; CHCl₃/MeOH=9/1).

The folate-containing peptidyl fragment Pte-Glu-(AA)_(n)-Cys-OH (9) isprepared by a polymer-supported sequential approach using theFmoc-strategy on an acid-sensitive Fmoc-Cys(Trt)-Wang resin (7), asshown in Scheme 14. R₁ is Fmoc, R₂ is Trityl, and DIPEA isdiisopropylethylamine. PyBop is applied as the activating reagent toensure efficient coupling. Fmoc-protecting groups are removed after eachcoupling step under standard conditions. Appropriately protected aminoacid building blocks, such as Fmoc-Glu-OtBu, N¹⁰-TFA-Pte-OH, and thelike, are used, as described in Scheme 14, and represented by in step(b) by Fmoc-AA-OH. Thus, AA refers to any amino acid starting material,that is appropriatedly protected. The coupling sequence (steps (a) &(b)) involving Fmoc-AA-OH is performed “n” times to preparesolid-supported peptide 8, where n is an integer and may equal 0 toabout 100. Following the last coupling step, the remaining Fmoc group isremoved, and the peptide is sequentially coupled to a glutamatederivative (step (c)), deprotected, and coupled to TFA-protected pteroicacid (step (d)). Subsequently, the peptide is cleaved from the polymericsupport upon treatment with trifluoroacetic acid, ethanedithiol, andtriisopropylsilane (step (e)). These reaction conditions result in thesimultaneous removal of the t-Bu, t-Boc, and Trt protecting groups. TheTFA protecting group is removed upon treatment with base (step (f)) toprovide the folate-containing Cys-containing peptidyl fragment 9.

Drug conjugates are prepared by reacting folate derivative 9 (0.9-0.95eq.) with drug thiosulfonate 6 in deionized water (0.04 M, pH adjustedto 7 with 0.1 N NaHCO₃) under argon for about 30 minutes, forming adisulfide bond. Upon evaporation of the methanol in vacuo, the conjugatemay be purified by preparative HPLC (Prep Novapak HR C18 19×300 mMcolumn; mobile phase (A)—1.0 mM phosphate buffer, pH=6; organic phase(B)—acetonitrile; conditions-gradient from 99% A and 1% B to 50% A and50% B in 30 minutes, flow rate=15 mL/minute).

EXAMPLE 14a

¹H NMR (DMSO-d₆) δ 4.7 (d, 1H), 4.95 (t, 1H), 6.7 (d, 4H), 6.9 (t, 1H),7.95 (d, 2H), 8.1 (d, 2H), 8.2 (m, 1H), 8.3 (s, 1H), 8.4 (s, 1H), 8.7(s, 1H), 10.2 (s, 1H), 11.8 (d, 2H).

EXAMPLE 14b

ES MS (m−H)⁻ 1436.4, (m+H)⁺ 1438.3.

EXAMPLE 14c

¹H NMR DMSO-d₆/D₂O) δ 1.0 (s, 1H), 1.1 (s, 1H), 1.6 (s, 1H), 1.8 (s,1H), 2.1 (s, 1H), 2.25 (s, 3H), 2.65 (dd, 2H), 3.7 (d, 1H), 4.4 (t, 1H),4.55 (q, 2H), 4.6 (d, 2H), 4.95 (d, 1H), 5.9 (t, 1H), 6.15 (s, 1H), 6.6(d, 2H), 7.85 (d, 2H), 7.95 (d, 2H), 8.6 (s, 1H), 8.95 (d, 1H).

EXAMPLE 14d

¹H NMR (DMSO-d₆/D₂O) δ 1.0 (s, 1H), 1.1 (s, 1H), 1.65 (s, 1H), 2.1 (s,1H), 2.25 (s, 3H), 2.6 (dd, 2H), 3.25 (dd, 1H), 3.6 (t, 2H), 3.7 (d,1H), 4.4 (t, 1H), 4.6 (d, 1H), 4.95 (d, 1H), 5.9 (t, 1H), 6.2 (s, 1H),6.6 (d, 2H), 7.7 (t, 1H), 7.9 (d, 2H), 7.95 (d, 2H), 8.6 (s, 1H), 9.1(d, 2H).

EXAMPLE 14e

¹H NMR (DMSO-d₆/D₂O) δ 10.85 (d, 2H), 1.05 (d, 2H), 1.2 (d, 2H), 1.7 (d,2H), 3.95 (d, 1H), 4.05 (dd, 1H), 5.4 (dd, 1H), 5.7 (dd, 1H), 6.65 (d,2H), 7.6 (d, 2H), 7.95 (s, 1H), 8.65 (s, 1H).

EXAMPLE 14f

ES MS (m+H)⁺ 1487.23; ¹H NMR (DMSO-d₆/D₂O) δ 0.9 (t, 2H), 1.3 (t, 2H),2.15 (t, 2H), 3.2 (dd, 1H), 4.0 (t, 1H), 4.15 (q, 1H), 5.3 (s, 2H), 5.5(s, 2H), 6.6 (d, 2H), 7.0 (s, 1H), 7.4 (m, 2H), 7.55 (d, 2H), 8.0 (d,2H), 8.6 (s, 1H).

Examples 14a, 14b, 14c, 14d, 14e, and 14f were prepared by the followinggeneral procedure. To a well stirred solution of the corresponding drughaving an —OH group (1 eq. in dry CH₂Cl₂ or dry THF) was added underargon 6-(trifluoromethyl)benzotriazolyl 2-(2′-pyridyldithioethylcarbonate (1.3 eq.) and NN-dimethylaminopyridine (1.5 eq.). The reactionmixture was stirred for 3 h, and the pyridyldithio-derivatized drug wasisolated by silica chromatography (>65% for each example). Thecorrepsonding peptidyl fragment (0.5 eq.), prepared according to thegeneral approach outlined in Scheme 12, was dissolved in DMSO. To theresulting clear yellow solution was added the pyridyl-dithio derivatizeddrug. After 30 min, the reaction was completed and the conjugatepurified by HPLC. In the case of Example 14e, the peptidyl fragmentPte-Glu-Asp-Arg-Asp-Asp-Cys-OH was first dissolved in water, and the pHof the solution was adjusted to 2.5 with 0.1 N HCl, causing the peptidylfragment to precipitate. The peptidyl fragment was collected bycentrifugation, dried, and dissolved in DMSO for subsequent reactionwith the pyridyldithio-derivatized drug.

EXAMPLE 15

The intermediate 4-(2-pyridinyldithio)benzylcarbonate of SN 38(10-hydroxy-7-ethylcamptothecin) was prepared according to the proceduredescribed by P. Senter et al., J. Org. Chem. 1990, 55, 2875, thedisclosure of which is incorporated herein by reference. The peptidylfragmant Pte-Glu-Asp-Arg-Asp-Cys-OH was dissolved in DMSO, and to theresulting clear yellow solution was added the pyridyldithio derivatizeddrug. After 30 min, the reaction was completed and the conjugatepurified by HPLC; ES MS (m+H)⁺ 1425.38; ¹H NMR (DMSO-d₆/D₂O) δ 0.9 (t),1.15 (t), 3.9 (t), 4.0 (t), 4.25 (t), 5.1 (m), 5.2 (s), 5.4 (s), 6.55(d), 7.25 (d), 7.35 (d), 7.5 (d), 7.9 (d), 8.55 (s).

EXAMPLE 16a

EXAMPLE 16b

The compounds of Examples 16a and 16b were prepared from the peptidylfragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH , prepared according to thegeneral procedure described in Scheme 12. The Michael addition of thispeptidyl fragment to the maleimido derivative of seco-CBI-bis-indoleresulted in the folate conjugates Example 16a. The peptidyl fragmentalso reacted with either the thiosulfonate or pyridyldithio-activatedvinblastine to form Example 16b. The maleimido derivative ofseco-CBI-bis-indole, and the thiosulfonate and pyridyldithio-activatedvinblastine intermediates were prepared using the procedures describedherein for other examples.

EXAMPLE 17a

Deacetylvinblastine monohydrazide (1 eq.) (see Barnett et al., J. Med.Chem., 1978, 21, 88, the disclosure of which is incorporated herein byreference) was treated in fresh distilled THF with 1 eq. oftrifluoroacetic acid. After stirring for 10 min the solution was treatedwith 1.05 eq. of N-(4-acetylphenyl)maleimide. Acyl hydrazone formationwas completed in 45 min and the solvent was evaporated. The peptidylfragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH (0.85 eq.), prepared accordingto the general approach outlined in Scheme 12, was dissolved in water,and the pH was adjusted to 2.5 with 0.1 N HCl, causing the peptide toprecipitate. The peptidyl fragment was collected by centrifugation,dried, and dissolved in DMSO. To the resulting clear yellow solution wasadded Hünig's base (15 eq.) and the acyl hydrazone Micahel adduct. After1 h, the final conjugate was purified by HPLC.

EXAMPLE 17b

EXAMPLE 17c

Examples 17b and 17c were prepared according to the procedure describedin Example 17a with the corresponding peptidyl fragments andmonohydrazide derivatives of CBI.

The compounds of Examples 18-41 were prepared according to the proceduregenerally described in Example 13. Examples 18-41 were characterized byelectrospray mass spectroscopy (ES MS), and other spectroscopictechniques, including 1D and 2D NMR, and UV.

EXAMPLE 18

ES MS (m+H)⁺ 1071.9, (m+Na)⁺ 1093.9; ¹H NMR (D₂O) δ 2.6 (t, 4H), 2.7 (t,4H), 4.15 (s, 2H), 5.45 (s, 2H), 7.75 (d, 2H), 8.15 (d, 2H), 8.9 (s,1H).

EXAMPLE 19

UV (nm) 233 (max), 255, 280; ¹H NMR (D₂O, NaOD, CD₃CN) δ 1.15 (d, 3H),2.3 (s, 3H), 3.6 (s, 1H), 3.85 (s, 3H), 4.9 (s, 1H), 5.3 (s, 1H), 6.5(d, 2H), 7.3 (m, 1H), 7.5 (d, 2H), 7.65 (d, 2H), 8.4 (s, 1H).

EXAMPLE 20

ES MS (m−H)⁻ 935.6, (m+H)⁺ 937.4, (m+Na)⁺ 959.5.

EXAMPLE 21

¹H NMR (D₂O, NaOD, CD₃CN) δ 0.1 (s, 1H), 1.1 (s, 3H), 1.2 (s, 3H), 1.75(s, 3H), 1.9 (s, 3H), 2.05 (s, 3H), 2.35 (s, 3H), 3.3 (dd, 2H), 3.8 (d,1H), 4.3 (q, 2H), 4.9 (d, 1H), 5.1 (d, 1H), 5.4 (q, 1H), 5.55 (d, 1H),5.65 (d, 1H), 6.1 (t, 1H), 6.35 (s, 1H), 6.9 (d, 2H), 7.9 (d, 2H), 8.15(d, 2H), 8.7 (s, 1H).

EXAMPLE 22

EXAMPLE 23

ES MS (m−H)⁻ 1136.5.

EXAMPLE 24

ES MS (m−H)⁻ 1136.3, (m+H)⁺ 1138.0.

EXAMPLE 25

ES MS (m+H)⁺ 1382.3, (m+Na)⁺ 1405.4.

EXAMPLE 26

ES MS (m−H)⁻ 1379.2, (m+H)⁺ 1381.2.

EXAMPLE 27

ES MS (mH)⁻ 949.2; ¹H NMR (D₂O) δ 1.55 (s, 3H), 1.95 (m, 2H), 2.05 (s,3H), 2.45 (s, 3H), 2.75 (dd, 2H), 2.95 (dd, 2H), 3.05 (s, 3H), 3.3 (dd,2H), 3.35 (d, 2H), 3.45 (t, 2H), 4.85 (q, 2H), 6.5 (d, 2H), 7.45 (d,2H), 8.5 (s, 1H).

EXAMPLE 28

¹H NMR (DMSO-d₆) δ 1.5 (s), 2.25 (t), 2.75 (m), 3.9 (q), 4.6 (d), 4.85(t), 6.6 (d), 7.6 (d), 7.9 (d), 8.15 (d), 8.25 (t), 8.65 (s), 8.7 (m),9.3 (m), 10.2 (t).

EXAMPLE 29

ES MS (m−H)⁻ 1413.5, (m+H)⁺ 1415.3.

EXAMPLE 30

ES MS (m+H)⁺ 1530.2; ¹H NMR (DMSO-d₆/D₂O) δ 1.2 (s, 1H), 2.9 (t, 1H),3.65 (t, 1H), 4.15 (t, 1H), 4.25 (t, 1H), 4.35 (t, 1H), 6.7 (d, 2H), 7.0(s, 1H), 8.1 (d, 2H), 8.25 (s, 1H),8.7 (s, 1H).

EXAMPLE 31

¹H NMR (DMSO-d₆) δ 1.75 (s, 1H), 1.85 (s, 1H), 2.1 (t, 2H), 4.3 (t, 1H),4.6 (d, 1H), 4.9 (t, 1H), 6.6 (d, 2H), 8.15 (s, 2H), 8.6 (s, 1H).

EXAMPLE 32

ES MS (m+H)⁺ 1408.4.

EXAMPLE 33

ES MS (m−H)⁻ 1491.1, (m+H)⁺ 1493.1; ¹H NMR (DMSO-d₆/D₂O) δ 4.15 (q, 1H),4.6 (d, 1H), 4.9 (t, 1H), 6.6 (d, 2H), 7.25 (s, 1H), 7.4 (d, 1H), 7.9(d, 1H), 7.95 (d, 2H), 8.15 (d, 2H), 8.6 (s, 1H).

EXAMPLE 34

¹H NMR (DMSO-d₆/D₂O) δ 2.1 (t, 2H), 2.75 (q, 2H), 4.3 (t, 1H), 4.65 (d,1H), 4.9 (t, 1H), 6.6 (d, 2H), 7.9 (d, 1H), 8.0 (d, 2H), 8.2 (t, 2H),8.6 (s, 1H).

EXAMPLE 35

EXAMPLE 36

ES MS (m+H)⁺ 1680.4; ¹H NMR (DMSO-d₆/D₂O) δ 0.3 (s, 3H), 0.35 (s, 3H),1.05 (s, 9H), 2.15 (t, 2H), 4.15 (t, 1H), 4.85)t, 1H), 6.6 (d, 2H), 7.55(t, 4H), 7.9 (d, 1H), 8.0 (s, 1H), 8.05 (d, 1H), 8.15 (s, 1H), 8.6 (s,1H).

EXAMPLE 37

¹H NMR (DMSO-d₆/D₂O) δ 1.1 (s, 3H), 1.8 (s, 1H), 4.55 (d, 1H), 4.8 (t,1H), 6.6 (d, 2H), 7.8 (d, 1H), 8.1 (d, 1H), 8.15 (s, 1H), 8.6 (s, 1H).

EXAMPLE 38

EXAMPLE 39

EXAMPLE 40

EXAMPLE 41

EXAMPLE 42 Inhibition of Tumor Growth in Mice Treated with EC112

The anti-tumor activity of the compounds of Examples 9b (EC111) and 9c(EC112), where the drug is daunorubicin, when administered intravenously(i.v.) to tumor-bearing animals, was evaluated in Balb/c mice bearingsubcutaneous M109 tumors. Four days post tumor inoculation in thesubcutis of the right axilla with 1×10⁶ M109 cells, mice (5/group) wereinjected i.v. twice a week for 4 weeks with 2-10 μmol/kg of the compoundof either Example 9b or Example 9c or with unconjugated daunorubicin orPBS. Tumor growth was measured using calipers at 3-day or 4-dayintervals in each treatment group. Tumor volumes were calculated usingthe equation V=a×b²/2, where “a” is the length of the tumor and “b” isthe width expressed in millimeters. Animal body weight was alsodetermined at 3-day or 4-day intervals.

As shown in FIGS. 1 and 2, treatment with the compound of Example 9c waseffective in delaying the growth of M109 tumors with no apparenttoxicity (based on animal body weights). Unconjugated doxorubicin alsoprovided an anti-tumor response, but with concomitant toxicity based onbody weights.

EXAMPLE 43 Inhibition of Tumor Growth in Mice Treated with EC105

The protocol was as described in Example 42 except that the compound ofExample 10a (EC105) was used, where the drug is bis-indolyl-seco-CBI.The compound of Example 10a was injected at a dose of 0.3 μmol/kg. Also,two subcutaneous tumor models were tested including the M109 model(folate receptor-positive) and the 4T1 model (folate receptor-negative),and in some animals a 67-fold excess of free folate (20 μmol/kg; FA) wascoinjected with the conjugate (i.e., the compound of Example 10a).

A striking anti-tumor response was observed with the compound of Example10a with no apparent toxicity based on animal body weights (see FIGS. 3and 4). The anti-tumor response with the compound of Example 10a wasblocked with excess free folate demonstrating the specificity of theresponse (see FIG. 3). As shown in FIG. 5, no anti-tumor activity wasobserved in the 4T1 model (folate receptor-negative) again demonstratingthe specificity of the response.

EXAMPLE 44 Inhibition of Tumor Growth in Mice Treated with EC145

The anti-tumor activity of the compound of Example 16b (EC145), wherethe drug is deacetylvinblastine monohydrazide, when administeredintravenously (i.v.) to tumor-bearing animals, was evaluated in Balb/cmice bearing subcutaneous M109 tumors. Approximately 11 days post tumorinoculation in the subcutis of the right axilla with 1×10⁶ M109 cells(average tumor volume at t_(o)=60 mm³), mice (5/group) were injectedi.v. two times a week (BIW), for 3 weeks with 1500 nmol/kg of EC145 orwith an equivalent dose volume of PBS (control). Tumor growth wasmeasured using calipers at 2-day or 3-day intervals in each treatmentgroup. Tumor volumes were calculated using the equation V=a×b²/2, where“a” is the length of the tumor and “b” is the width expressed inmillimeters.

As shown in FIG. 6, treatment with EC145 was effective in delaying thegrowth of M109 tumors relative to growth of M109 with tumors insaline-treated animals.

EXAMPLE 45 Inhibition of Tumor Growth in Mice Treated with EC140

The anti-tumor activity of the compound of Example 17a (EC140), wherethe drug is deacetylvinblastine monohydrazide, when administeredintravenously (i.v.) to tumor-bearing animals, was evaluated in Balb/cmice bearing subcutaneous M109 tumors. Approximately 11 days post tumorinoculation in the subcutis of the right axilla with 1×10¹⁶ M09 cells(average tumor volume at t_(o)=60 mm³), mice (5/group) were injectedi.v. three times a week (TIW), for 3 weeks with 1500 nmol/kg of EC140 orwith an equivalent dose volume of PBS (control). Tumor growth wasmeasured using calipers at 2-day or 3-day intervals in each treatmentgroup. Tumor volumes were calculated using the equation V=a×b²/2, where“a” is the length of the tumor and “b” is the width expressed inmillimeters.

As shown in FIG. 7, treatment with EC140 was effective in delaying thegrowth of M109 tumors relative to growth of M109 with tumors insaline-treated animals.

EXAMPLE 46 Inhibition of Tumor Growth in Mice Treated with EC136

The anti-tumor activity of the compound of Example 10b (EC136), wherethe drug is CBI, when administered intravenously (i.v.) to tumor-bearinganimals, was evaluated in DBA mice bearing subcutaneous L1210A tumors.Approximately 5 days post inoculation of 0.25×10⁵ L1210A cells into thesubcutis of the right axilla (average tumor volume at t_(o)˜50 mm³; mice5/group) animals were injected i.v. three times a week (TIW) for 3 weekswith 400 nmol/kg of the EC136 or an equivalent dose volume of PBS alone(control). Tumor growth was measured using calipers at 2-day or 3-dayintervals in each treatment group. Tumor volumes were calculated usingthe equation V=a×b²/2, where “a” is the length of the tumor and “b” isthe width expressed in millimeters.

As shown in FIG. 8, treatment with EC136 was effective in delaying thegrowth of L1210A tumors, relative to growth of L1210A tumors insaline-treated animals.

EXAMPLE 47 Inhibition of Cellular DNA Synthesis by Various Folate-DrugConjugates

The compounds of Examples 17b, 10b, 16a, 10c, 17a, 16b, 14e, and 15(EC135, EC136, EC137, EC138, EC140, EC145, EC158, and EC159,respectively) were evaluated using an in vitro cytotoxicity assay thatpredicts the ability of the drug to inhibit the growth of folatereceptor-positive KB cells. The compounds were comprised of folatelinked to a respective chemotherapeutic drug, as prepared according tothe protocols described herein. The KB cells were exposed for up to 7 hat 37 ° C. to the indicated concentrations of folate-drug conjugate (seex-axes of graphs shown in FIGS. 9-16) in the absence or presence of atleast a 100-fold excess of folic acid. The cells were then rinsed oncewith fresh culture medium and incubated in fresh culture medium for 72hours at 37° C. Cell viability was assessed using a ³H-thymidineincorporation assay.

As shown in FIGS. 9-16) dose-dependent cytotoxicity was measurable, andin most cases, the IC₅₀ values (concentration of drug conjugate requiredto reduce ³H-thymidine incorporation into newly synthesized DNA by 50%)were in the low nanomolar range. Furthermore, the cytotoxicities ofthese conjugates were reduced in the presence of excess free folic acid,indicating that the observed cell killing was mediated by binding to thefolate receptor.

Similar results were obtained in this type of assay using EC158 and celllines including IGROV (known cell line), A549-Clone-4 (A549 cellstransfected with human folate receptor cDNA), New Line-01 (Line-01 cellmutant selected in vivo for folate receptor expression), M109, 4T1Clone-2 (4T1 cells transfected with murine folate receptor cDNA), andHeLa cells.

1. A vitamin receptor binding drug delivery conjugate comprising: (a) avitamin receptor binding moiety; (b) a bivalent linker; and (c) a drug,or an analog or derivative thereof; wherein the vitamin receptor bindingmoiety is covalently linked to the bivalent linker; the drug, or theanalog or the derivative thereof, is covalently linked to the bivalentlinker; and the bivalent linker comprises one or more componentsselected from the group consisting of spacer linkers, releasablelinkers, and heteroatom linkers, and combinations thereof; providingthat the bivalent linker includes at least one releasable linker that isnot a disulfide.
 2. The drug delivery conjugate of claim 1 wherein thevitamin receptor binding moiety is selected from the group consisting ofvitamins, and vitamin receptor binding analogs and derivatives thereof.3. The drug delivery conjugate of claim 1 wherein the heteroatom linkeris a nitrogen, oxygen, or sulfur atom, or is selected from the group offormulae consisting of —NHR¹NHR²—, —SO—, —S(O)₂—, and —NR³O—, whereinR¹, R², and R³ are each independently selected from the group consistingof hydrogen, alkyl, aryl, arylalkyl, substituted aryl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, and alkoxyalkyl.
 4. Thedrug delivery conjugate of claim 1 wherein the spacer linker is selectedfrom the group consisting of carbonyl, thionocarbonyl, alkylene,cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl,sulfonylalkyl, alkylenesulfoxylalkyl, alkylenesulfonylalkyl,carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl,1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of saidspacer linkers is optionally substituted with one or more substituentsX¹; wherein each substituent X¹ is independently selected from the groupconsisting of alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino,aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl,sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl, carboxy,carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, and R⁷-acylaminoalkyl,wherein R⁴ and R⁵ are each independently selected from the groupconsisting of an amino acid, an amino acid derivative, and a peptide,and wherein R⁶ and R⁷ are each independently selected from the groupconsisting of an amino acid, an amino acid derivative, and a peptide. 5.The drug delivery conjugate of claim 4 wherein the heteroatom linker isnitrogen, and wherein the substituent X¹ and the heteroatom linker aretaken together with the spacer linker to which they are bound to form anheterocycle.
 6. The drug delivery conjugate of claim 5 wherein theheterocycle is selected from the group consisting of pyrrolidines,piperidines, oxazolidines, isoxazolidines, thiazolidines,isothiazolidines, pyrrolidinones, piperidinones, oxazolidinones,isoxazolidinones, thiazolidinones, isothiazolidinones, and succinimides.7. The drug delivery conjugate of claim 1 wherein the releasable linkeris selected from the group consisting of methylene, 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,haloalkylenecarbonyl, alkylene(dialkylsilyl), alkylene(alkylarylsilyl),alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl,(diarylsilyl)aryl, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonylalkyl,iminoalkylidenyl, carbonylalkylidenimninyl, iminocycloalkylidenyl,carbonylcycloalkylideniminyl, alkylenesulfonyl, alkylenethio,alkylenearylthio, and carbonylalkylthio, wherein each of said releasablelinkers is optionally substituted with one or more substituents X²;wherein each substituent X² is independently selected from the groupconsisting of alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino,aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl,sulfhydrylalkyl, alkylthioalkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl, carboxy,carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, and R⁷-acylaminoalkyl,wherein R⁴ and R⁵ are each independently selected from the groupconsisting of an amino acid, an amino acid derivative, and a peptide,and wherein R⁶ and R⁷ are each independently selected from the groupconsisting of an amino acid, an amino acid derivative, and a peptide. 8.The drug delivery conjugate of claim 7 wherein the heteroatom linker isnitrogen, and wherein the substituent X² and the heteroatom linker aretaken together with the releasable linker to which they are bound toform an heterocycle.
 9. The drug delivery conjugate of claim 8 whereinthe heterocycle is selected from the group consisting of pyrrolidines,piperidines, oxazolidines, isoxazolidines, thiazolidines,isothiazolidines, pyrrolidinones, piperidinones, oxazolidinones,isoxazolidinones, thiazolidinones, isothiazolidinones, and succinimides.10. The drug delivery conjugate of claim 1 wherein the heteroatom linkeris nitrogen, and wherein the releasable linker and the heteroatom linkerare taken together to form a divalent radical comprisingalkyleneaziridin-1-yl, alkylenecarbonylaziridin-1-yl,carbonylalkylaziridin-1-yl, alkylenesulfoxylaziridin-1-yl,sulfoxylalkylaziridin-1-yl, sulfonylalkylaziridin-1-yl, oralkylenesulfonylaziridin-1-yl, wherein each of said releasable linkersis optionally substituted with one or more substituents X²; wherein eachsubstituent X² is independently selected from the group consisting ofalkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl,alkylaminoalkyl, dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,alkylthioalkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, carboxy, carboxyalkyl,alkyl carboxylate, alkyl alkanoate, guanidinoalkyl, R⁴-carbonyl,R⁵-carbonylalkyl, R⁶-acylamino, and R⁷-acylaminoalkyl, wherein R⁴ and R⁵are each independently selected from the group consisting of an aminoacid, an amino acid derivative, and a peptide, and wherein R⁶ and R⁷ areeach independently selected from the group consisting of an amino acid,an amino acid derivative, and a peptide.
 11. The drug delivery conjugateof claim 10 wherein the heteroatom linker is nitrogen, and thereleasable linker and the heteroatom linker are taken together to form adivalent radical comprising alkyleneaziridin-1-yl,carbonylalkylaziridin-1-yl, sulfoxylalkylaziridin-1-yl, orsulfonylalkylaziridin-1-yl,.
 12. The drug delivery conjugate of claim 11wherein the spacer linker is selected from the group consisting ofcarbonyl, thionocarbonyl, alkylenecarbonyl, cycloalkylenecarbonyl,carbonylalkylcarbonyl, and 1-(carbonylalkyl)succinimid-3-yl, whereineach of said spacer linkers is optionally substituted with one or moresubstituents X¹; wherein each substituent X¹ is independently selectedfrom the group consisting of alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide, and wherein R⁶ and R⁷ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide; and wherein the spacer linker is bonded to the releasablelinker to form an aziridine amide.
 13. The drug delivery conjugate ofclaim 1 wherein the drug is a mitomycin, a mitomycin derivative, or amitomycin analog, and the releasable linker is selected from the groupconsisting of carbonylalkylthio, carbonyltetrahydro-2H-pyranyl,carbonyltetrahydrofuranyl,1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of saidreleasable linkers is optionally substituted with one or moresubstituents X², wherein each substituent X² is independently selectedfrom the group consisting of alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide, and wherein R⁶ and R⁷ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide; and wherein the aziridine of the mitomycin is bonded tothe releasable linker to form an acylaziridine.
 14. The drug deliveryconjugate of claim 1 wherein the drug includes a double-bonded nitrogenatom, wherein the releasable linker is selected from the groupconsisting of alkylenecarbonylamino and1-(alkylenecarbonylamino)succinimid-3-yl, and wherein the releasablelinker is bonded to the drug nitrogen to form an hydrazone.
 15. The drugdelivery conjugate of claim 1 wherein the drug includes a sulfur atom,the releasable linker is selected from the group consisting ofalkylenethio and carbonylalkylthio, and wherein the releasable linker isbonded to the drug sulfur to form a disulfide.
 16. The drug deliveryconjugate of claim 4 wherein the vitamin receptor binding moiety isfolate which includes a nitrogen, and the spacer linker is selected fromthe group consisting of alkylenecarbonyl, cycloalkylenecarbonyl,carbonylalkylcarbonyl, and 1-(carbonylalkyl)succinimid-3-yl, wherein thespacer linker is bonded to the folate nitrogen to form an imide or analkylamide.
 17. The drug delivery conjugate of claim 16 wherein eachsubstituent X¹ is independently selected from the group consisting ofalkyl, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, carboxy, carboxyalkyl,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide, and wherein R⁶ and R⁷ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide.
 18. The drug delivery conjugate of claim 4 wherein theheteroatom linker is nitrogen, and the spacer linker is selected fromthe group consisting of alkylenecarbonyl, cycloalkylenecarbonyl,carbonylalkylcarbonyl, and 1-(carbonylalkyl)succinimid-3-yl, whereineach of said spacer linkers is optionally substituted with one or moresubstituents X¹ and the spacer linker is bonded to the nitrogen to forman amide.
 19. The drug delivery conjugate of claim 4 wherein theheteroatom linker is sulfur, and the spacer linker is selected from thegroup consisting of alkylene and cycloalkylene, wherein each of saidspacer linkers is optionally substituted with carboxy, and the spacerlinker is bonded to the sulfur to form a thiol.
 20. The drug deliveryconjugate of claim 4 wherein the heteroatom linker is sulfur, and thespacer linker is selected from the group consisting of1-alkylenesuccinimid-3-yl and 1-(carbonylalkyl)succinimid-3-yl, and thespacer linker is bonded to the sulfur to form a succinimid-3-ylthiol.21. The drug delivery conjugate of claim 7 wherein the heteroatom linkeris oxygen, and the releasable linker is selected from the groupconsisting of methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene,1-alkoxyalkylenecarbonyl, and 1-alkoxycycloalkylenecarbonyl, whereineach of said releasable linkers is optionally substituted with one ormore substituents X², and the releasable linker is bonded to the oxygento form an acetal or ketal.
 22. The drug delivery conjugate of claim 7wherein the heteroatom linker is oxygen, and the releasable linker ismethylene, wherein said methylene is substituted With anoptionally-substituted aryl, and the releasable linker is bonded to theoxygen to form an acetal or ketal.
 23. The drug delivery conjugate ofclaim 7 wherein the drug includes a nitrogen atom, the heteroatom linkeris nitrogen, and the releasable linker is selected from the groupconsisting of carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl,carbonyl(biscarboxyaryl)carbonyl, and the releasble linker is bonded tothe heteroatom linker nitrogen to form an amide, and also bonded to thedrug nitrogen to form an amide.
 24. The drug delivery conjugate of claim7 wherein the drug includes an oxygen atom, the heteroatom linker isnitrogen, and the releasable linker is selected from the groupconsisting of carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl,carbonyl(biscarboxyaryl)carbonyl, and the releasable linker is bonded tothe heteroatom linker nitrogen to form an amide, and also bonded to thedrug oxygen to form an ester.
 25. The drug delivery conjugate of claim 7wherein the heteroatom linker is nitrogen, and the releasable linker isselected from the group consisting of iminoalkylidenyl,carbonylalkylideniminyl, iminocycloalkylidenyl, andcarbonylcycloalkylideniminyl, wherein each of said releasable linkers isoptionally substituted with one or more substituents X², and thereleasable linker is bonded to the nitrogen to form an hydrazone. 26.The drug delivery conjugate of claim 7 wherein the heteroatom linker isoxygen, and the releasable linker is selected from the group consistingof alkylene(dialkylsilyl), alkylene(alkylarylsilyl),alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, and(diarylsilyl)aryl, wherein each of said releasable linkers is optionallysubstituted with one or more substituents X², and the releasble linkeris bonded to the oxygen to form a silanol.
 27. The drug deliveryconjugate of claim 1 wherein the drug includes a nitrogen atom, and thereleasable linker is haloalkylenecarbonyl, optionally substituted withone or more substituents X²; wherein each substituent X² isindependently selected from the group consisting of alkyl, alkoxy,alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl,substituted heteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkylalkanoate, guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino,and R⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide, and wherein R⁶ and R⁷ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide; and the releasable linker is bonded to the drug nitrogento form an amide.
 28. The drug delivery conjugate of claim 1 wherein thedrug includes an oxygen atom, and the releasable linker isalkyleneoxycarbonyl or haloalkylenecarbonyl, optionally substituted withone or more substituents X²; wherein each substituent X² isindependently selected from the group consisting of alkyl, alkoxy,alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl,substituted heteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkylalkanoate, guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino,and R⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide, and wherein R⁶ and R⁷ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide; and the releasable linker is bonded to the drug oxygen toform a carbonate or an ester.
 29. The drug delivery conjugate of claim 1wherein the heteroatom linker is oxygen, the spacer linker is1-alkylenesuccinimid-3-yl, optionally substituted with one or moresubstituents X¹, and the releasable linker is selected from the groupconsisting of methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene,1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, wherein each ofsaid releasable linkers is optionally substituted with one or moresubstituents X²; wherein each substituent X² is independently selectedfrom the group consisting of alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide, and wherein R⁶ and R⁷ are each independently selectedfrom the group consisting of an amino acid, an amino acid derivative,and a peptide; and wherein the spacer linker and the releasable linkerare each bonded to the heteroatom linker to form a succinimid-1-ylalkylacetal or ketal.
 30. The drug delivery conjugate of claim 1 wherein thebivalent linker comprises an heteroatom linker, a spacer linker, and areleasable linker taken together to form3-thiosuccinimid-1-ylalkyloxymethyloxy, where the methyl is optionallysubstituted with alkyl or substituted aryl.
 31. The drug deliveryconjugate of claim 1 wherein the bivalent linker comprises an heteroatomlinker, a spacer linker, and a releasable linker taken together to form3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl forms anacylaziridine with the drug, or analog or derivative thereof.
 32. Thedrug delivery conjugate of claim 1 wherein the bivalent linker comprisesan heteroatom linker, a spacer linker, and a releasable linker takentogether to form 1-alkoxycycloalkylenoxy.
 33. The drug deliveryconjugate of claim 1 wherein the bivalent linker comprises a spacerlinker, an heteroatom linker, and a releasable linker taken together toform alkyleneaminocarbonyl(dicarboxylarylene)carboxylate.
 34. The drugdelivery conjugate of claim 1 wherein the bivalent linker comprises areleasable linker, a spacer linker, and a releasable linker takentogether to form dithioalkylcarbonylhydrazide, where the hydrazide formsan hydrazone with the drug, or analog or derivative thereof.
 35. Thedrug delivery conjugate of claim 1 wherein the bivalent linker comprisesan heteroatom linker, a spacer linker, and a releasable linker takentogether to form 3-thiosuccinimid-1-ylalkylcarbonylhydrazide, where thehydrazide forms an hydrazone with the drug, or analog or derivativethereof.
 36. The drug delivery conjugate of claim 1 wherein the bivalentlinker comprises an heteroatom linker, a spacer linker, an heteroatomlinker, a spacer linker, and a releasable linker taken together to form3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where thedisubstituted silyl is substituted with alkyl or optionally substitutedaryl.
 37. The drug delivery conjugate of claim 1 wherein the bivalentlinker comprises a plurality of spacer linkers selected from the groupconsisting of the naturally occurring amino acids and stereoisomersthereof.
 38. The drug delivery conjugate of claim 1 wherein the bivalentlinker comprises a releasable linker, a spacer linker, and a releasablelinker taken together to form 3-dithioalkyloxycarbonyl, where thecarbonyl forms a carbonate with the drug, or analog or derivativethereof.
 39. The drug delivery conjugate of claim 1 wherein the bivalentlinker comprises a releasable linker, a spacer linker, and a releasablelinker taken together to form 3-dithioarylalkyloxycarbonyl, where thecarbonyl forms a carbonate with the drug, or analog or derivativethereof, and the aryl is optionally substituted.
 40. The drug deliveryconjugate of claim 1 wherein the bivalent linker comprises an heteroatomlinker, a spacer linker, a releasable linker, a spacer linker, and areleasable linker taken together to form3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the alkylideneforms an hydrazone with the drug, or analog or derivative thereof, eachalkyl is independently selected, and the oxyalkyloxy is optionallysubstituted with alkyl or optionally substituted aryl.
 41. The drugdelivery conjugate of claim 1 wherein the bivalent linker comprises areleasable linker, a spacer linker, and a releasable linker takentogether to form 3-dithioalkyloxycarbonylhydrazide.
 42. The drugdelivery conjugate of claim 1 wherein the bivalent linker comprises areleasable linker, a spacer linker, and a releasable linker takentogether to form 3-dithioalkylamino, where the amino forms a vinylogousamide with the drug, or analog or derivative thereof.
 43. The drugdelivery conjugate of claim 42 wherein the alkyl is ethyl.
 44. The drugdelivery conjugate of claim 1 wherein the bivalent linker comprises areleasable linker, a spacer linker, and a releasable linker takentogether to form 3-dithioalkylaminocarbonyl, where the carbonyl forms acarbamate with the drug, or analog or derivative thereof.
 45. The drugdelivery conjugate of claim 44 wherein the alkyl is ethyl.
 46. The drugdelivery conjugate of claim 1 wherein the bivalent linker comprises areleasable linker, a spacer linker, and a releasable linker takentogether to form 3-dithioarylalkyloxycarbonyl, where the carbonyl formsa carbamate or a carbamoylaziridine with the drug, or analog orderivative thereof.
 47. A pharmaceutical composition comprising a drugdelivery conjugate of claim 1, and a pharmaceutically acceptablecarrier, diluent, or excipient therefor.
 48. A method of eliminating apopulation of pathogenic cells in a host animal harboring the populationof pathogenic cells wherein the members of the pathogenic cellpopulation have an accessible binding site for a vitamin, or an analogor a derivative thereof, and wherein the binding site is uniquelyexpressed, overexpressed, or preferentially expressed by the pathogeniccells, said method comprising the step of administering to said host adrug delivery conjugate of claim 1, or a pharmaceutical compositionthereof.
 49. A vitamin receptor binding drug delivery conjugateintermediate comprising: (a) a vitamin receptor binding moiety; (b) abivalent linker, having a first end and a second end; and (c) a couplinggroup; wherein the coupling group is a nucleophile, an electrophile, ora precursor thereof, capable of forming a covalent bond with a drug, oran analog or derivative thereof; the vitamin receptor binding moiety iscovalently attached to the bivalent linker at the first end of thebivalent linker, and the coupling group is covalently attached to thebivalent linker at the second end of the bivalent linker; and thebivalent linker comprises one or more components selected from the groupconsisting of spacer linkesr, releasable linkesr, and heteroatomlinkers, and combinations thereof; providing that the bivalent linkerincludes at least one releasable linker that is not a disulfide.
 50. Theintermediate of claim 49 wherein the vitamin receptor binding moiety isa vitamin, or a vitamin receptor binding analog or derivative thereof.51. A vitamin receptor binding drug delivery conjugate having theformula:V-L-Dwherein L is selected from the group consisting of (l_(s))_(a),(l_(H))_(b), and (l_(r))_(c), and combinations thereof; where (l_(r)) isa releasable linker, (l_(s)) is a spacer linker, and (l_(H)) is anheteroatom linker; V is a vitamin receptor binding moiety, and D is adrug, or an analog or a derivative thereof; and a, b, and c are eachindependently 0, 1, 2, 3, or 4; providing that L includes at least one(l_(r)) that is not a disulfide.
 52. A vitamin receptor binding drugdelivery conjugate having the formula:V-L-Dwherein L is selected from the group consisting of (l_(s))_(a) and(l_(H))_(b), and combinations thereof, where (l_(s)) is a spacer linkerand (l_(H)) is an heteroatom linker; and a and b are each independently0, 1, 2, 3, or 4; and V is a vitamin receptor binding binding moiety,and D is a drug, or an analog or a derivative thereof.
 53. The drugdelivery conjugate of claim 52 wherein the drug is a hapten.
 54. Thedrug delivery conjugate of claim 53 wherein the hapten is selected fromthe group consisting of fluorescein and dinitrophenyl.
 55. Apharmaceutical composition comprising a drug delivery conjugate of claim1, and a pharmaceutically acceptable carrier, diluent, or excipienttherefor.
 56. A method of eliminating a population of pathogenic cellsin a host animal harboring the population of pathogenic cells whereinthe members of the pathogenic cell population have an accessible bindingsite for a vitamin, or an analog or a derivative thereof, and whereinthe binding site is uniquely expressed, overexpressed, or preferentiallyexpressed by the pathogenic cells, said method comprising the step ofadministering to said host a drug delivery conjugate of claim 1, or apharmaceutical composition thereof.
 57. A vitamin receptor binding drugdelivery conjugate intermediate comprising: (a) a bivalent linker,having a first end and a second end; (b) a drug, or an analog or aderivative thereof, and (c) a coupling group; wherein the bivalentlinker comprises one or more components selected from the groupconsisting of spacer linkers, releasable linkers, and heteroatomlinkers, and combinations thereof; providing that the bivalent linkerincludes at least one releasable linker that is not a disulfide; thecoupling group is a nucleophile, an electrophile, or a precursorthereof, capable of forming a covalent bond with a vitamin receptorbinding moiety; the coupling group is covalently attached to thebivalent linker at the first end of the bivalent linker, and the drug,or analog or derivative thereof is covalently attached to the bivalentlinker at the second end of the bivalent linker.
 58. The intermediate ofclaim 57 wherein the vitamin receptor binding moiety is a vitamin, or avitamin receptor binding analog or derivative thereof; the couplinggroup is a Michael acceptor; and the bivalent linker includes areleasable linker having a formula selected from the group consisitingof —C(O)NHN═, —NHC(O)NHN═, and —CH₂C(O)NHN═.
 59. The intermediate ofclaim 57 wherein the bivalent linker comprises a first releasablelinker, and a first spacer linker; and the first releasable linker, thefirst spacer linker, and the coupling group are taken together to form acompound having the formula:

where D is the drug, or an analog or a derivative thereof, and n is aninteger selected from the group consisting of 1, 2, 3, and
 4. 60. Theintermediate of claim 59 wherein the vitamin receptor binding moietyincludes an alkylthiol nucleophile.
 61. A process for preparing acompound having the formula:

where V is a vitamin receptor binding moiety; L is selected from thegroup consisting of (l_(r))_(c), (l_(s))_(a), and (l_(H))_(b), andcombinations thereof, where (l_(r)) is a releasable linker, (l_(s)) is aspacer linker, (l_(H)) is an heteroatom linker, and a, b, and c areintegers selected from the group consisting of 0, 1, 2, 3, and 4; and Dis a drug, or an analog or a derivative thereof; comprising: (a)reacting a compound having the formula:

with a compound having the formula:

and (b) forming a hydrazone derivative of the drug, or the analog orderivative thereof.
 62. A process for preparing a compound having theformula:

where V is a vitamin receptor binding moiety; L is selected from thegroup consisting of (l_(r))_(c), (l_(s))_(a), and (l_(H))_(b), andcombinations thereof, where (l_(r)) is a releasable linker, (l_(s)) is aspacer linker, (l_(H)) is an heteroatom linker, and a, b, and c areintegers selected from the group consisting of 0, 1, 2, 3, and 4; and Dis a drug, or an analog or a derivative thereof; comprising: reacting acompound having the formula:

with a compound having the formula:


63. The process of claim 62 wherein the drug or the analog or derivativethereof is selected from the group consisting of daunomycins,daunomycinones, daunorubicins, aclamycins, taxols, chromomycins, andaurilic acids.